Enzyme Kinetics and Inhibition in Neurotransmitter Metabolism

Abstract

In this review, the major steps of the neurotransmitter lifecycle are explored through the kinetics of the enzymes that facilitate them, with an emphasis on how inhibitors can affect these kinetics. To develop effective treatment plans for neuropsychiatric illnesses, it is important to understand the precise mechanisms by which inhibitors impact enzyme activity and, in turn, neurotransmitter levels. To obtain data on enzyme kinetics and inhibition mechanisms, a thorough review methodology is used to analyze original research articles, review papers, and experimental studies. Data was obtained from multiple experimental designs, such as pharmacological evaluations, kinetic studies, and in vitro assays The literature was surveyed to identify research articles that specifically characterized the relevant enzymes’ kinetic properties through in vitro and in vivo studies. Additionally, we reviewed articles that investigated these. enzymes’ inhibition and characterized the drug molecules’ kinetic properties and their effect on their targets This presents how inhibitors of the enzymes that metabolize neurotransmitters can dramatically change their kinetics, which can impact signaling and neurotransmitter availability. Although using inhibitors to modify enzyme activity may have therapeutic advantages, side effects, and interactions must be carefully considered. This work emphasizes the potential of targeted inhibition as a therapeutic strategy and the significance of enzyme kinetics in neurotransmitter metabolism. It is advised that more research be done to determine the long-term consequences of enzyme inhibition and how it affects mental and neurological health. While comparing standard enzyme kinetics to inhibitor kinetics, certain inhibitors are more effective than others, as they exhibit tighter binding and lower IC50 values, which reflects their greater potency. In contrast to the native substrate, these selective inhibitors significantly reduce the enzyme’s activity, while less effective inhibitors fail to achieve this degree of inhibition.

Introduction

Neurotransmitters (NTs) are chemical messengers that enable neurons to send signals to each other throughout the body to help it perform its everyday functions like moving limbs and sensing the environment¹. The body uses a variety of neurotransmitters, such as acetylcholine for contracting muscles, learning, and memory² and for regulating sleep, mood, digestion, and blood clotting.

Understanding neurotransmitters’ role and metabolism is essential to comprehending the complex mechanisms that control the neurophysiological system. The synthesis, release, reuptake, and degradation of neurotransmitter molecules are major steps in the life cycle of neurotransmitters and are important for the regulation and proper function of neurotransmitters³.

The synthesis of neurotransmitters is the process by which primary metabolic molecules, such as amino acids, are converted to neurotransmitters. Synthesis pathways can involve multiple enzymatic steps, which can all be regulated and influence the levels of neurotransmitter⁴.

The discharge of neurotransmitters from neurons occurs in response to an external stimulus and is known as neurotransmitter release. The release of neurotransmitters occurs at synapses, which are locations where various nerve impulses are transmitted⁵.

The reabsorption of certain neurotransmitters by a neurotransmitter transporter is known as neurotransmitter reuptake. Because it facilitates neurotransmitter recycling and regulates the concentration of neurotransmitters in the synapse, reuptake plays a crucial role in synaptic physiology by influencing the duration of signals originating from neurotransmitter release⁴.

Degradation of neurotransmitters occurs when an enzyme modifies or breaks them down so that their receptors cannot bind them. This prevents signaling from occurring and allows the product to be recycled back into the neurotransmitter metabolism⁶.

Disturbances in the homeostasis of neurotransmitters resulting from abnormalities and deficiencies in metabolism can cause altered neurotransmission-mediated signaling, which can be correlated with several different neurological and neurodegenerative diseases including schizophrenia, amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease (AD)⁷.

Drugs can likewise affect the homeostasis of neurotransmitters and alter how neurons communicate with each other by interacting with enzymes and proteins involved in neurotransmitter metabolism⁸. Because drugs can specifically alter the various steps in the neurotransmitter life cycle, they can be utilized in the treatment of these diseases and disorders. Therefore, a deeper understanding of the activity of enzymes in the neurotransmitter lifecycle, and how they interact with their substrates and these drugs, is important for developing treatments⁸.

Given the important role that enzymes play in neurotransmitter metabolism, understanding the kinetics of these enzymes becomes crucial. Altering the kinetics of enzyme-catalyzed reactions can disrupt neurotransmitter homeostasis, influencing everything from synthesis to degradation. Insights into these kinetic parameters can help clarify how changes in enzyme activity contribute to neurophysiological disorders and interactions with inhibitors

Enzymes are biological catalysts that speed up reactions in living organisms; enzymes catalyze reactions by binding to reactant molecules called substrates reducing the activation energy and increasing the reaction rate9 The rate of the reaction catalyzed by enzymes is described by its kinetics. These kinetics measurements can be used to define an enzyme’s activity, allowing us to study and observe when and how their activity is altered.

The fundamental way of modeling and understanding enzyme kinetics was first defined by Michaelis and Menten over 100 years ago and provides a platform for understanding enzymes.  The Michaelis-Menten equation defines that the rate of an enzyme-catalyzed reaction depends on the concentration of the enzyme and its substrate⁹.

There are three important constants when studying enzyme kinetics: k_cat, V_max, and K_M. k_cat is the turnover rate, the number of substrate molecules that are being converted to a product by a single enzyme molecule.⁹.

 The Michaelis constant, K_M, is the substrate concentration at which the enzymes achieve half of the maximum velocity of the enzyme. V_max (maximum velocity) is the highest theoretical rate for an enzyme-catalyzed reaction⁹.

Measuring these kinetic parameters allows researchers to observe altered enzyme activity, such as reduced turnover rates or changes in substrate binding affinity, which can indicate the effects of inhibitors or mutations on enzyme function. In addition to their native or natural substrate, enzymes can also bind to other molecules.

In addition to their native or natural substrate, enzymes can also bind to other molecules. Inhibitors are molecules that can alter enzymes’ natural kinetics. Enzyme inhibitors are molecules that interact in some way, either permanently or temporarily, with enzymes to either decrease the rate of an enzyme-catalyzed reaction or stop enzymes from functioning normally¹⁰.

The inhibition constant (Ki) is a measure of the affinity of an inhibitor for its target enzyme, the concentration at which the inhibitor achieves half maximum inhibition, and it can be compared to the Michaelis constant (Km) to evaluate how effectively an inhibitor competes with the substrate

The IC₅₀ is one of the parameters measuring how different inhibitors interact with their target enzyme; IC₅₀ is the concentration of an inhibitor required to decrease the rate of an enzymatic reaction by 50%¹¹. This parameter is often used in conjunction with kcat, the turnover rate, to assess how well an inhibitor affects the enzyme’s catalytic efficiency and overall activity

Competitive, uncompetitive, and mixed-type are the three major modalities of enzyme-inhibitor interaction. Competitive inhibition refers to the mutually exclusive binding of the inhibitor and substrate, which is generally same-site binding. When an inhibitor preferentially binds to an enzyme form primed by substrate binding, this is known as uncompetitive inhibition. In a mixed-type inhibition instance, the inhibitor and substrate may attach to the enzyme separately, with the inhibitor’s concentration influencing the substrate’s ability to bind either favorably or unfavorably¹¹. Using inhibitors as drugs to alter the kinetics of specific enzymes is a common way to treat diseases. Drugs such as antacids can alter enzymes by increasing the pH and changing the kinetics of these enzymes¹². Specifically for neurotransmitters, many inhibitors act upon the enzymes involved in the neurotransmitter lifecycle.  

This review will discuss and further define the steps of neurotransmitter metabolism and some of the enzymes involved, specifically how the altered activity of these enzymes can cause different effects in the body, and examine some of the ways these targets can be drugged. Gaining insight into some of the most influential enzymes involved in neurotransmitter recycling, how altering their activity can cause effects and which strategies and targets in medicine are useful for treating diseases are the objectives of this review.

The most effective targets for inhibition, the importance of specific versus nonspecific inhibition, the importance of the mechanisms by which the enzymes can be targeted and inhibited, and how the challenges of specific enzymes affect the outcomes have been identified and discussed in this review.

Results

Synthesis 

There are multiple enzymes involved in synthesizing neurotransmitters. Some well-known and studied enzymes involved in this process are tryptophan hydroxylase 2 (TPH2) and choline acetyltransferase (ChAT), which are involved in the synthesis of synthesizing  the neurotransmitters serotonin and acetylcholine, respectively.

Tryptophan hydroxylase. Tryptophan hydroxylase 2 (TPH2) is involved in synthesizing the neurotransmitter serotonin found in vertebrates. This enzyme is the rate-determining step in the pathway for producing serotonin and therefore has the greatest impact on the levels of this neurotransmitter, making it the best target for drugging.  TPH2 is an isozyme of tryptophan hydroxylase; an isozyme is one of two or more enzymes with identical functions but different structures.TPH2 regulates serotonin levels and appears in the brain’s serotonergic neurons¹³.          

Tidemand et al. evaluated the kinetics of the catalytic domain of the human tryptophan hydroxylase (chTPH). The kinetic parameters for chTPH1 and chTPH2 are presented in Table 1,2 were found to be k_cat =  258±20 min^-1 and K_M = 13.1±1.3 μM, while tryptophan hydroxylase 1 (chTPH1) kinetic values were k_cat =108 ± 10 min^-1 and K_M = 13.1±1.3 μM

Enzyme	Kcat (min-1))	KM (μM)
chTPH2	258±20	13.1±1.3
chTPH1	108 ± 10	13.1±1.3 μM

The K_M values of wild-type chTPH1 and chTPH2  were identical, but  chTPH2 was shown to have a ~2-fold higher k_cat than chTPH1. Additionally, only chTPH1 showed substrate inhibition. These kinetic parameters have a good correlation with the values previously reported by Windahl et al. and Moran et al¹⁴¹⁵

earlier findings, however, occasionally there are small differences. This could be because the data are derived from TPH in various organisms. Further, TPH2 has been reported not to be inhibited by its substrates but has also been previously reported to exhibit inhibition nonetheless, albeit with relatively high inhibition constants (418 – 970 μM).

Para-chlorophenylalanine (pCPA) and para-ethynylphenylalanine are both inhibitors of TPH2. pCPA is the most wideldy used TPH2 inhibitor, with over 300 different citations of its inhibiton of TPH2. The Ki reported was 300 uM in a rat brain TPH2 assay¹⁶ In vivo administration of pCPA leads to a prolonged reduction in brain levels of 5-HT  in laboratory rodents and a single administration of pCPA reduces TPH2 protein density in the rat brain. This drug has notable limitations, including a delayed onset of effect , the need for relatively high doses, and limited efficacy. Roughly 10 injections of pCPA (300 mg/kg, ip) are required to achieve an 80% reduction in brain 5-HT levels. Additionally, pCPA lacks specificity, inhibiting both TPH2 and TPH1¹⁶.To address these limitations of pCPA, the para-position chloride was replaced with an ethynyl functional group, resulting in the synthesis of a new drug: para-ethynylphenylalanine (pEPA). In vitro, pEPA Ki was reported 32.6 uM, and pEPA was reported that a single administration of pEPA (30 mg/kg, ip) significantly reduces 5-HT levels and TPH2 activity in the rat brain for 24 hours¹⁶. pEPA had a lower Ki value than pCPA with pEPA Ki = 32.6 uM and pCPA Ki = 300 uM. This indicates that pEPA hads a higher affinity for its target than pCPA, as reflected by its lower Ki value. 

Inhibitor	Ki (uM)
Para-chlorophenylalanine	300
para-ethynyl phenylalanine	32.6

Choline acetyltransferase. Another enzyme that is involved in the synthesis of another neurotransmitter is choline acetyltransferaseChAT. Choline acetyltransferase (ChAT) is the most important enzyme that participates in acetylcholine synthesis and is currently the most accurate indicator for tracking the functional state of cholinergic neurons in the central and peripheral nervous systems. 

In many cholinergic neurons, ChAT is widely distributed intraneurally and can be found in cell bodies, dendrites, axons, and axon terminals¹⁷.

Ryan et al. used ChAT  choline acetyltransferase from rat cerebrum and bovine caudate nuclei to determine the K_M which are shown in Table 3. For choline, K_M was reported as 1010 μM for the rat cerebrum and 714 μM for bovine caudate, and for the co-substrate acetyl-CoA (AcCoA) the K_M values were reported as 46.5 μM for the rat cerebrum and 16.5 μM for bovine caudate. The Michaelis constants for choline were calculated by secondary replots of the initial velocity data¹⁸.

Substrate	Cell	KM  (μM)
choline	rat cerebrum	1010
choline	bovine caudate nuclei	714
acetyl-CoA (AcCoA)	rat cerebrum	46.5
acetyl-CoA (AcCoA)	bovine caudate nuclei	16.5

Omeprazole, lansoprazole, and pantoprazole are some inhibitors of choline acetyltransferase (ChAT )¹⁹.  These molecules, which are also proton-pump inhibitors (PPIs), selectively inhibit choline acetyltransferase (ChAT) inhibiting neuronal signaling within the brain, possibly be related to the increased risk of dementia²⁰

.Baidya et al. shared that it can be challenging to interpret IC₅₀ values in some situations because they can change based on factors like the concentration of the enzyme substrates. As a result, the authors carried out thorough enzyme-inhibition kinetics analyses that took into account various substrate and medication concentrations. In this manner, it was possible to ascertain the most dependable inhibition constants (K_i) and how PPIs function as ChAT inhibitors. Omeprazole, lansoprazole, and pantoprazole exhibited IC₅₀ values of 0.1, 1.5, and 5.3 μM.²¹.

The estimated K_i for omeprazole ranged between 0.07 and 0.14 μM.  Non-linear enzyme-ligand kinetic analyses found a high probability (>99%) to suggest that this PPI functions as a mixed-competitive reversible inhibitor of ChAT with choline concentration. This mode of operation hads the significant consequence of ensuring that omeprazole binds to the enzyme with high affinity, irrespective of the enzyme’s state. It also implieds that as would be expected from a compound that functions as a full competitive inhibitor, the endogenous in vivo choline concentration may only slightly impair this compound’s inhibition of ChAT.

The K_i for omeprazole is was lower than the reported K_M of rat ChAT  (1010 μM), and for bovine ChAT (714 μM ) the K_i is was around 1000 times smaller than the K_M. A smaller K value suggests that  omeprazole was found to be is more effective at binding to the enzyme than its native substrate

For ChAT, rabeprazole’s K_i value was 0.0175 μM and tenatoprazole was 0.018 μM. Furthermore, both exhibited non-competitive reversible ChAT inhibitory properties. A non-competitive mode of action suggests that rabeprazole and tenatoprazole can inhibit ChAT regardless of the in vivo concentration of its substrate, choline. They both have a similar high affinity for the free and choline-bound enzymes²⁰.

Inhibitors	Type of Inhibitor	IC50 (μM)	Ki (μM)
Omeprazole	Irreversible/selective	0.1	–
Lansoprazole	Irreversible	1.5	–
Pantoprazole	Irreversible	0.14	0.07 – 0.14
Rabeprazole	Reversible	–	0.0175
Tenatiorazole	Reversible	–	0.018

Rabeprazole’s and tenatoprazole’s K_i values were very similar with  only a 0.005 difference from each other. Similar to omeprazole their K_i values were lower than ChAT K_M values showing that all inhibitors have a greater binding affinity. But Rabeprazole had the smallest K_i value and omeprazole had the largest at 0.07 to 0.14 μM. Comparing these inhibitors, Rabeprazole was the most effective at binding to the enzyme. 

Reuptake

The reuptake of neurotransmitters is an essential component of neurotransmitter metabolism and is mediated by a variety of enzymes. The serotonin transporter (SERT) and the dopamine transporter (DAT) are both crucial enzymes that are responsible for the reuptake of these important neurotransmitters. 

Dopamine transporter. Dopamine transporter (DAT) is an enzyme responsible for dopamine reuptake and is part of a superfamily of neurotransmitter transporters dependent on  Na⁺/Cl⁻²². DAT is located within the membrane of various nerve cells in the brain and drives the reuptake of extracellular transmitters into presynaptic neurons, which in turn regulates the temporal and spatial dynamics of dopamine (DA) neurotransmission²².   Abnormal DA levels are linked to several disorders, including Parkinson’s disease, depression, and bipolar disorder, suggesting that DAT may have a role in the genesis of these conditions²². 

Ross previously described the kinetics of the dopamine transporter DAT in vitro in the mouse striatum.  The K_M value of the initial uptake of [³H]DA into synaptosomes from the mouse striatum was 0.209 +- 0.02 μM, where the V_max was 0.007 ± 0.00129 μM/g tissue/min which are shown in Table 5. (Ross et al. 1991). Other authors have used these kinetic parameters and the methods for their determination to study the effect of various inhibitors on DAT and how they impact its activity. 

Enzyme	KM   (μM)	Vmax ( μM/g tissue/min)
Dopamine Transporter	0.209 +- 0.02 μM	0.007 ± 0.00129

Cocaine and methamphetamine are both inhibitors of the dopamine transporter (DAT )²³’²⁴. Cocaine can increase dopamine signaling by blocking DAT. Methamphetamine increases synaptic dopamine levels many times above baseline by inhibiting DAT-mediated dopamine uptake and stimulating dopamine efflux. Methamphetamine also decreases wound healing and increases susceptibility to infections by targeting peripheral immune cells that express DAT²⁵.

Jones et al. investigated the dopamine release and reuptake by DAT in the mouse striatum and the effect of amphetamine on its kinetics. Using electrical stimulation on these brain slices evoked increased measured DA concentration, which rapidly returneds to regular steady state constant. The results of these studies are reported in Table 6.   The rate constant for uptake before amphetamine, defined as the ratio of V_max to K_M under saturated conditions, was found to be  20 ± 2  s^-1 In the presence of 10 µM amphetamine, uptake was significantly slowed, and the rate constant was determined to be 0.10 ± 0.06 s^-1. Before amphetamine was added the concentration of DA, [DA] p, was 9μM, V_max was recorded as 3.50 μM/s, and K_M as 0.16 μM. Thirty minutes after 10 μM of amphetamine was added the values recorded were:  [DA]p = 2.7 μM, V_max = 3.5 μM/s and K_M = 8.0 μM. Thisese data indicated that dopamine transport rates were significantly lowered by amphetamine²⁶.

Han and Gu, compared the potency of Amphetamine on DAT when inhibiting a mouse brain and found that the Ki value was approximately 0.6 μM.

DAT concerning Amphetamine	Vmax (μM/s)	KM    (μM)	[DA]p (μM)
Before Amphetamine	3.50	0.16	9
30 minutes After 10 μM Amphertamine	3.5	8	2.7

The function of DAT can be described by a simple transporter model and in general, drug interactions can be explained by a few basic concepts. DAT can move dopamine bidirectionally, but outward transport mainly depends on the availability of inwardly oriented transporters. The role of transporters is to move the neurotransmitter from the synapse to inside the cell. However, these enzymes can also move neurotransmitters from inside the cell to out if they are already oriented inside the cell. Amphetamine causes can cause DAT to release rather than uptake dopamine by binding to the transporter in the synapse and bringing it inside the cell. Once DAT is oriented inside the cell, the transporter can return dopamine to the synapse. Amphetamine is a broad-spectrum inhibitor and can inhibit many enzymes that are part of the reuptake step in the metabolism of neurotransmitters. 

Conversely, cocaine inhibits DAT by blocking the transporter completely, which is a different mechanism from amphetamine, which inverts the direction of transport²⁷.             

Ferris et al. researched the effect of self-administered cocaine on rat brains and found that the application of cocaine on the dopamine transporter DAT greatly increased the apparent K_M value of DAT. The results of these studies are reported in Table 7 The max binding of dopamine to the transporter with cocaine was  28.75 ± 3.14 μM, while the natural K_M of the dopamine transporter was  209 ± 20 nM  (0.2 μM)., which is an order of magnitude difference between the K_M with cocaine²⁷.          

DAT concerning Cocaine	KM  (μM)
Native binding of DAT	0.2
max binding of Dopamine to DAT the transporter with cocaine	28.75 ± 3.14

A larger K_M value indicateds that dopamine isn’t wasn’t binding as well with the transporter in the presence of cocaine. Cocaine was found to reduce the binding of substrate to the transporter, and the more often the rat subjects self-administered the cocaine the less of an effect it had.  Cocaine’s ability to inhibit the enzyme is was reduced over time, which is important in drug addiction wherein subjects then do more cocaine to achieve the same effect. It was also found that cocaine did not affect the ability of amphetamine to then inhibit the enzyme, which shows these drugs affect DAT by two different mechanisms. 

Before cocaine inhibited DAT, the enzyme KM value was 0.2μM. After the inhibition of cocaine, the DAT KM value increased to 28.75 ± 3.14 μM, a greater than 100-fold increase. Before the inhibition of amphetamine, the enzyme Km value was 0.16 μM. After the inhibition of cocaine, the DAT KM value increased to 8 μM. Cocaine significantly increased the DAT KM value, indicating a stronger inhibitory effect on DAT affinity compared to amphetamine. Therefore, cocaine appears to more drastically impact DAT function, potentially leading to more pronounced alterations in dopamine signaling.

Serotonin transporter. Another enzyme that is part of the reuptake of a different neurotransmitter is serotonin transporter (SERT), which reuptakes serotonin. SERT is an essential membrane protien exhibited in neurons of the raphe nuclei in the brain. The main function of SERT is the quick clearing of serotonin from the synaptic cleft, and causing the termination of the chemical signal transduction in serotonergic neurons. Additionally, SERT has an important role in functions such as sleeping, mood, and appetite. SERT is the primary target for selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs), prescribed for depression and anxiety disorders²⁸.

Kilic et al. have previously examined the kinetics of human serotonin transporter by measuring the transmigration rate over time in two cell lines, human-derived HeLa cells and primate-derived COS-7 cells which are presented in Table 8.²⁹. In the HeLa BS cell line, the average transportation rate of SERT was 0.131 ± 0.01 pmol/min/mg, the K_M recorded was 1.28 ± 0.19 μM, and V_max recorded as 1.73 ± 0.06 pmol/min/mg32. In the COS-7 cells, these values were given as Transportation rate of SERT = 0.416 ± .007 pmol/min/mg, K_M = 1.04 ± 0.51 μM, and V_max = 1.37 ± 0.56 pmol/min/mg.

Type of cell line	KM         (μM)	Vmax (pmol/min/mg)	Average Transportation Rate (pmol/min/mg)
Human-derived HeLa cells	1.28 ± 0.19	1.73 ± 0.06	0.131 ± 0.01
Primate-derived COS-7 cells	1.04 ± 0.51	1.37 ± 0.56	0.416 ± .007

Some inhibitors of serotonin transporter (SERT) are ​​selective serotonin uptake inhibitors (SSRIs) such as S-citalopram (S-CIT), sertraline, fluoxetine, and paroxetine. SSRIs are used to treat different types of disorders like depression and anxiety²⁸. The inhibitory kinetics of some of these inhibitors are outlined in Table 9.  Citalopram hydrobromide is a selective serotonin reuptake inhibitor. The FDA primarily indicates citalopram hydrobromide for the treatment of adult depression. By increasing serotonergic activity in the central nervous system, citalopram hydrobromide has an antidepressant effect. Citalopram hydrobromide was found to have an IC₅₀ of 0.0018 μM³⁰. and has a high selectivity and affinity to serotonin transporters with a K_i of 0.001939 μM³¹.The K_M found for SERT previously was  1.28 ± 0.19 μM for the HeLa BS cell line and the K_M = 1.04 ± 0.51 μM for  COS-7. The K_i of Citalopram hydrobromide is orders of magnitude smaller than the K_M of SERT for serotonin, which indicates a strong binding affinity between the inhibitor and the enzyme and shows that the inhibitor has a higher affinity for SERT than the substrate. 

Fluoxetine, a racemic mixture of the stereoisomers R– and S-fluoxetine, is another selective serotonin reuptake inhibitor (SSRI) that regulators have approved for use in psychiatry for various clinical uses³¹’³².

Inhibitor	Ki   (μM)	IC50 (μM)
Citalopram hydrobromide	0.001939	0.0018
Fluoxetine	0.19119	5.992
Amphetamine	20 – 40	—–

Fluoxetine was observed to have a K_i = 0.19119 μM³³. and has an IC₅₀ of 5.992 μM. Similarly to citalopram hydrobromide, fluoxetine had s a smaller K_i than the K_M of SERT which shows that its it was better at binding to the enzyme then the native substrate serotonin. Fluoxetine was shown to have a higher IC₅₀ than citalopram hydrobromide, which means that citalopram hydrobromide is was more effective at inhibiting SERT as a lower IC₅₀ indicates a higher potency.

Amphetamine also inhibits SERT, in addition to DAT, where the Ki value reported by Dawn D Han, Howard H Gu, was 20 to 40 μM.  While amphetamine inhibits both the serotonin transporter (SERT) and the dopamine transporter (DAT), it displays significantly higher affinity for DAT. The Ki value for amphetamine’s inhibition of DAT is approximately 0.6 μM, which was around 30 to 60 times more than its Ki value for SERT inhibition (20 to 40 μM), as reported by Han and  Gu. This difference suggests that amphetamine’s primary effect is likely mediated through DAT inhibition, with a comparatively weaker impact on SERT.³⁴.

Citalopram had a lower Ki value than Fluoxetine with Citalopram Ki = 0.001939 μM and Fluoxetine Ki =  0.19119 μM. This indicates that citalopram had a higher affinity for its target than fluoxetine, as reflected by its lower Ki value.  Consequently, citalopram was likely to be a more potent inhibitor in this context than fluoxetine. Fluoxetine was shown to have a higher IC50 than citalopram hydrobromide, which means that citalopram hydrobromide was more effective at inhibiting SERT as a lower IC50 indicates a higher potency.

Degradation 

Degradation of neurotransmitters is the final step and an important part of neurotransmitter metabolism. Multiple different enzymes are responsible for the degradation of different neurotransmitters. Monoamine oxidase A (MAO-A) and acetylcholinesterase AChE are two of the most significant enzymes.

Monoamine oxidase A.  Monoamine oxidase A (MAO-A) is an important enzyme that is part of the degradation of multiple different monoaminergic neurotransmitters and plays an important role in their neurotransmission regulation³⁵’³⁶.

 Monoamine oxidase (MAO) is an FAD-containing enzyme that facilitates the α-carbon oxidation of monoamines, such as serotonin, norepinephrine, and dopamine, which are all broken down by MAO-A³⁷.   Monoamine oxidase A (MAO-A) has an important role in brain development, as well as regulating mood, sleep,  and appetite³⁸. Together with its isoenzyme MAO-A, monoamine oxidase-B (MAO-B) is a FAD-dependent mitochondrial enzyme that catalyzes the oxidative deamination of structurally diverse amines³⁹. MAO-A contains multiple binding sites for a variety of substrates required for its activity, and its catalytic mechanism proceeds through multiple different steps throughout its reaction. As MAO-A  acts on a variety of different neurotransmitters the kinetic parameters will vary for each substrate, which are outlined in Table 10⁴⁰.

Substrate	KM Km (μM)	Vmax                                 (pmol min–1 mg protein–1),	Vmax/KM (μmol M–1 min–1 mg protein–1)
Adrenaline	125 ± 42	379 ± 54	3.03 ± 1.11
dopamine	212 ± 33	680 ± 123	3.21 ± .77
5-hydroxytryptamine	137 ± 24	228 ± 31	1.66 ± 0.37 4642

Some inhibitors of MAO-A are phenelzine, tranylcypromine, and isocarboxazid⁴⁰.  As MAO-A is responsible for removing norepinephrine, serotonin, and dopamine from the brain, MAOIs work non-selectively to decrease the degradation of these neurotransmitters, allowing them to have a more prolonged effect on their target receptors⁴¹.

Many different classes of drugs inhibit MAO-A through a variety of mechanisms. There are four main types of inhibition observed for MAO: reversible, irreversible, mechanism-based irreversible, and poor substrate. Each type of inhibition affects the enzyme differently, with different results. 

A subclass of MAOIs known as reversible inhibitors, selectively and reversibly inhibit the MAO-A enzyme. These reversible inhibitors primarily work by competitive inhibition. D-amphetamine is one of the most basic reversible inhibitors of MAO. It is used to treat attention deficit hyperactivity disorder (ADHD), although its pharmacology and clinical effects are complicated. It was has been shown to inhibits both MAO and monoamine reuptake systems. With a K_i value of 15 μM, it is was observed to be an effective reversible competitive inhibitor of MAO-A⁴²’⁴³. while the inhibition is mixed for MAO B.  When comparing the K_i value of D-amphetamine to the K_M values from the different substrates, the K_M values ranged from  125  to 212 μM, where the K_i of  D-amphetamine is 15 μM. This K_i is substantially smaller than the reported K_M values showing that D-amphetamine is more effective at binding with MAO-A than any of the above substrates listed. The  IC₅₀ is approximately ten times higher with MAO B because both reduced and oxidized MAO B are present during turnover. 

In contrast to reversible inhibition, irreversible inhibition of an enzyme results in the enzyme’s permanent deactivation.  To regain activity, MAO-inhibitor adducts must be eliminated and freshly synthesized MAO has to take their place. Following irreversible inhibition, MAO activity in rat brains was found to recover over a nine-day half-life⁴⁰. One of the main reasons irreversible MAOIs are effective medications is because of the slow regeneration of MAO in vivo. Suicide, time-dependent, enzyme-activated, covalent, and mechanism-based inhibitors are all terms used to describe different irreversible MAO inhibitors³⁹. The seven requirements for the designation—time dependence of inactivation, observation of saturation kinetics, substrate protection, irreversibility, fixed stoichiometry, involvement of an enzyme-catalyzed step, and inactivation before release of active species— have been described in detail by Silverman⁴⁴ Propargylamines, cyclopropylamines, hydrazines, aminoacetamides, and aryloxazolidinones are among the classes of irreversible MAO inhibitors.                         

The propargylamines are a particularly well-studied class of irreversible MAOIs. Different propargylamine inhibitors can be distinguished from one another not only by how well they bind to MAO but also by how quickly they render the enzyme inactive. Thus, the rate at which distinct propargylamines inactivate MAO A or MAO B provides information about the inhibitor’s selectivity⁴⁵. Clorgyline’s affinity for MAO-A is three orders of magnitude (>1000x) greater than that of the B-form, and its rate of inactivation (k_inact) for MAO-A is 0.76 min⁻¹, compared to 0.006 for MAO-B, which accounts for the inhibitor’s strong MAO-A selectivity.,  In contrast, L-deprenyl only demonstrates a 40-fold difference in the affinity of the two forms, and the rates of inactivation by L-deprenyl for MAO A and B are 0.14 and > 0.99 min⁻¹, respectively, which significantly increases the inhibitor selectivity towards the B-form.  Further, Pargyline is primarily non-selective because it only slightly increases the affinity for MAO B, but the inactivation rates for both forms are extremely close. In a single molecule, the new generation of propargylamine multi-target inhibitors for the treatment of Alzheimer’s disease contains moieties that inhibit both cholinesterase and MAO. One member of this class, ASS234, inhibits membrane-bound MAO-A with K_i and k_inact values of 0.053 μM and 0.133 min⁻¹, respectively⁴⁶.

Clorgyline is an irreversible  potent and selective monoamine oxidase (MAO) inhbitor, used for research purposes, particularly in inhibiting the growth of tumor cells and reducing the aggressiveness of prostate cancer⁴⁷  Clorgyline was reported to have Ki values of 0.054 and 58 μM and IC50 values of 0.0012 and 1.9 μM for MAO-A and MAO-B, respectively⁴⁸

Inhibitors	Kinact (1/min)		Ki (μM)
	MAO-A	MAO-B	—
propargylamines	0.76	0.006	—
L-deprenyl	0.14	> 0.99	—
ASS234	0.133	—	0.053
D-amphetamine	—	—	15

The K_i of propargylamine ASS234 is was smaller than the reported K_M values of MAO-A, similar to D-amphetamine where the inhibitors are were more effective at binding with MAO-A than the original substrates. Comparing the two inhibitors, the K_i for propargylamine is was 0.053 μM and the K_i of D-amphetamine is was 15 μM. Propargylamine hads a smaller K_i value which indicates that it hads a stronger binding affinity with MAO-A than D-amphetamine⁴⁹.

Acetylcholinesterase. Another enzyme that participates in the degradation of a different neurotransmitter is acetylcholinesterase (AChE). Acetylcholinesterase (AChE)  is a cholinergic enzyme that degrades acetylcholine (ACh), a naturally occurring neurotransmitter. AChE is found in postsynaptic neuromuscular junctions, muscles, and nerves. The main role of AChE is to cease neuronal communication and signaling between synapses to avert ACh dispersal and activation of nearby receptors⁵⁰. AChE also plays a role in neural development besides its common function of terminating neural transmission. 

The activity of AChe has been reported for many different organisms against the substrate acetylthiocholine. The K_M for rats was found to be 2650 μM and the K_M determined for a house fly was 1800 μMSome of these are displayed in Table 12.

Pohanka et al. determined the kinetics of the electrical eel acetylcholinesteraseAChE, using two different substrates: acetylthiocholine and indoxyl acetate. The values were determined to be, K_M as 206 μM and the V_max as  4.97 × 10^-7 mol/s for the acetylthiocholine⁵¹. This study was done to assess the inhibition by the inhibitor paraoxon and subsequent reactivation by several different oxine oxime inhibitors 

Organism/cell type	KM (μM)	Vmax (mol/s)
Rat	2650	–
House fly	1800	–
Electric Eel	206	4.97 × 10-7

AChE inhibitors, also known as anti-cholinesterases, prevent the cholinesterase enzyme from degrading ACh, thereby increasing the level and duration of neurotransmitter action. 

There are various inhibitors of AChE, such as nerve agents like sarin, and pesticides such as paraoxon and parathion⁵². AChE inhibitors can be classified into two groups based on their method of execution: irreversible and reversible. 

An essential function of reversible AChE inhibitors is the pharmacological control of the enzyme activity. These compounds, which have different functional groups have been used as an antidote for anticholinergic overdose as well as in the diagnosis and/or treatment of several diseases, including Alzheimer’s disease (AD), postoperative ileus, and glaucoma⁵³.

A selective and reversible inhibitor of AChE, donepezil binds to the peripheral anionic site to produce both symptomatic and causative effects in the treatment of AD by delaying the buildup of amyloid plaque. The primary therapeutic application of donepezil is in the palliative management of mild to moderate AD. Moreover, according to certain clinical trials, donepezil also enhances cognitive function in individuals exhibiting severe symptoms of AD. Donepezil was found to inhibit AChE with an IC₅₀ value of 53.6 ± 4.0 ng/mL. 

Since the brain synthesizes AChE slowly, irreversible inhibitors increase therapeutic benefits and lower the risk of peripheral toxicity. According to clinical studies, irreversible AChE inhibitors can be very successful and accepted⁵⁴.

A powerful acetylcholinesterase (AChE) irreversible inhibitor, diazoxon is a toxic byproduct of the pesticide diazinon. When diazinon passes through the body, it may undergo hepatic oxidation to produce diazoxon, which inhibits AChE at peripheral and central nervous system nerve terminals. Acetylcholine builds up at receptors as a result of this inhibition, causing cholinergic reactions in the nervous system and neuromuscular junctions. These reactions may result in particularly apparent symptoms in the gastrointestinal, cardiovascular, and respiratory systems⁵⁵.

Inhibitor	Ki (μM/L)	IC50 (ng/mL)
Donepezil	–	53.6 ± 4.0
Diaxonon	0.7899	0.053

Diaxonon’s IC₅₀ for AChE was found to be 0.053 μM, and its K_i =0.7899 μM/L,. MAO-AAChE was shown to have a K_M of 206 μM/L with the electrical eel, and when comparing the K_i of diazinon and the K_M of MAO-A, the K_i is smaller than the K_M, which indicates that diazinon binds better with the enzyme than does its substrate. The IC₅₀ of Diaxonon is also very low which shows that Diaxonon is very effective at inhibiting the enzyme. 

Discussion

Based on a survey of the various mechanisms of inhibition presented for neurotransmitter regulation, it was found that the enzymatic mechanisms of action could be associated with different of action dictated the most effective mechanisms of inhibition. It was observed that i Irreversible inhibitors tend to be morewere effective with enzymes involved in the synthesis and degradation pathways because these processes rely on covalent interactions between the enzymes and substrates. This is exemplified by the significantly smaller IC50 value of diaxonon (irreversible) versus donepezil (reversible) for AChE. These enzymes that follow covalent mechanisms appear to be are therefore more likely to be inhibited by reactive and irreversible inhibitors.  In contrast,  reuptake transporter enzymes often were found to interact with reversible inhibitors, which temporarily bind to the transporter before detaching via non-covalent interactions.

Every inhibitor ultimately affects the concentration of neurotransmitters in the synapse, but the mechanisms by which this occurs can vary. In the case of synthesis and degradation enzymes, inhibitors primarily work by blocking the enzyme’s function, preventing it from facilitating the chemical reactions it usually catalyzes. This inhibition of synthesizing enzymes results in decreased neurotransmitter concentration, while inhibition of degrading enzymes leads to an increase in their concentration. On the other hand, reuptake transporters can be targeted by inhibitors that may reverse the enzyme’s direction rather than merely blocking its activity. This is was found to be particularly relevant to the dopamine transporter, which can operate in two directions, offering different opportunities for inhibition to alter the direction of dopamine flux in the synapse.

It was also noted that certain enzymes involved in neurotransmitter metabolism were more selective than others, meaning that their inhibition could affect the flux of one or many different neurotransmitters

Synthesis enzyme inhibitors are usually more selective because these enzymes are highly specific to the neurotransmitter precursor they act upon. Similarly,It was found that serotonin reuptake transporter (SERT) inhibitors exhibited high specificity and selectivity because SERT is responsible for only the reuptake of serotonin.  This is why, inhibitors must be highly selective to effectively target only serotonin. Conversely, the dopamine transporter (DAT) moves and reuptakes multiple different molecules, making DAT inhibitors less selective specific compared to SERT inhibitors. DAT inhibitors need to affect a larger range of neurotransmitter targets due to the multiple molecules that DAT transports. This distinction highlights the varying degrees of specificity suggests that enzyme target selection could be important required for effective inhibition across different neurotransmitter systems.

Monoamine oxidases (MAOs), which degrade many neurotransmitters are similar to DAT. Inhibiting MAOs can change the actions of multiple neurotransmitters, therefore its inhibitors are less selective specific for regulating a single neurotransmitter. This broad activity spectrum of MAOs shows the importance of designing drugs that can modulatinge the enzyme’s action across various neurotransmitter pathways.

Many different inhibitors targeting enzymes function differently to inhibit the enzyme properly. Understanding how different enzymes work with neurotransmitters and inhibitor molecules is important when creating a drug and knowing how to affect the neurotransmitter’s concentration correctly. 

An analysis of K_M values across different enzymes reveals that choline acetyltransferase (ChAT) has a notably high K_M value, recorded at 1010 μM for the rat and 714 μM for bovine caudate. These values are significantly larger than those observed for other enzymes, indicating a potentially easier inhibition of ChAT. Interestingly, every inhibitor identified presented had a K_i value lower than the K_M, suggesting that ChAT might be more readily inhibited due to its high K_M values. This insight provides an intriguing perspective on enzyme kinetics and inhibition, particularly in the context of neurotransmitter synthesis and degradation.

In general, the complex relationship between neurotransmitter systems’ inhibitors and enzymes emphasizes how difficult pharmacological interventions can be. A thorough understanding of these interactions will are necessary to enable us to effectively customize therapeutic strategies to modulate neurotransmitter concentrations, ultimately advancing the development of medications intended to treat neurological disorders.

The findings of this study provide essential insights into the enzymatic regulation of different steps of neurotransmitter metabolism, contributing significantly to our understanding of neurochemical balance and potential therapeutic interventions. This review brings together many different enzymes that participate in the steps of neurotransmitter metabolism, identifying reported enzymes’ kinetic values and how inhibitors can alter those specific kinetics. More literature on enzymes in the neurotransmitter metabolism cycle and how different neurotransmitters can be affected by inhibitors within the cycle should be identified. Comparing different types of inhibitors and different enzymes can offer a pathway to refine different drugs for better outcomes. Different inhibitors can suggest a novel mechanism by which these drugs might impact signaling and associated neurological disorders. Sharing the different affinities and mechanisms of inhibition can underscore the need for personalized medicine approaches.

Identifying inhibitors and describing the enzymatic mechanisms underlying neurotransmitter regulation were the main goals of the study. By offering thorough presenting kinetic analyses and investigating the effects of different inhibitors on important enzymes involved in neurotransmitter metabolism, the study effectively achieved these goals. 

Some key areas for future research have been identified, such as f. Finding more selective specific MAO-A and MAO-B inhibitors to minimize side effects and enhance therapeutic efficacy for psychiatric and neurodegenerative disorders. This paper covered how different MAOIs were less selective specific because of their multiple targets such as substrates including serotonin, norepinephrine, tyramine, and dopamine for MAO-A and monoamine oxidaseMAO-B substrates dopamine and tyramine. Due to these differences in substrate profiles, identifying inhibitors specific to either MAO-A or MAO-B could be beneficial.

but looking to more selective inhibitors could be beneficial. Specifically, inhibitors that only targeted one MAO, A or B. MAO-A was seen to have better activity on serotonin, so targeting MAO-A with a more selective inhibitor will affect the concentration of serotonin more, similar to MAO-B and dopamine. Designing inhibitors that are more selective for MAO-A or MAO-B could be useful toA or B will allow you to be more specific and more selectively change regulate those neurotransmitter neurotransmitters.

Integrating kinetic data into personalized medicine approaches, considering individual variations in enzyme activity and inhibitor response for tailored treatment strategies is also encouraged as a target for further research. For instance, in, TThe isoform-specific substrate inhibition mechanism of human tryptophan hydroxylase, chTRP1/chTRP2 the authors made different mutations to those enzymes to observe their kinetic data and how it was different from the original chTRP1/chTRP2 values. Gathering kinetic data on other mutated enzymes would be of interest.                                           

. By presenting the kinetic data for the enzymes discussed and how their inhibitors altered these kinetics this paper shared a base for understanding the importance of enzyme kinetics and inhibition in neurotransmitter regulation Also explores the long-term effects of cocaine and methamphetamine on DAT to understand their contribution to addiction. Additionally, investigates the potential cross-reactivity of SSRIs with other neurotransmitter systems. As shown in this paper cocaine and methamphetamine had different effects on the kinetic values, but looking at a longer period how would the effects change and lead to addiction, with kinetic values?  

There were limitations within this review. The study draws from kinetic data derived from various organisms, enzyme constructs, and assay conditions, potentially affecting the generalizability of findings. Species-specific differences in enzyme activity may not fully represent human physiological conditions. There was variability in substrate binding and inhibition across different studies highlighting the need for standardized experimental conditions to ensure consistent results.

In conclusion, the way enzymes manage neurotransmitters is crucial for keeping our brains balanced. As we learn more about these processes, trying to make big advances in treating neurological issues while understanding the variety of competitive, noncompetitive, and uncompetitive inhibitors, and how they work to inhibit specific enzymes and do their jobs. Every enzyme works differently with its substrate or inhibitor; knowing and learning how these interactions work is crucial to improving and influencing proper treatment. This research is not just about science; it’s about understanding the core of how our brains work and how we feel. We should continue exploring this, as it could lead to new treatments that improve the lives of many people.

Methods

For this review, several databases, including PubMed and Google Scholar, were utilized to locate relevant research. To achieve thorough search coverage, a strategic combination of keywords and phrases, such as “enzyme kinetics” and “kinetic values,” enabled the extraction of the desired parameters. Using Peer-reviewed articles, conference papers, and research that directly addressed the research question. Studies with weak methodology, duplicate articles across multiple databases, and articles for which the full text was not available were omitted. Further, only English-language studies were taken into account to avoid translation biases and guarantee consistency in the review process.

 Numerous study designs, including observational studies, experimental studies, qualitative research, and systematic reviews, are discussed in this review. A more thorough understanding of the research topic is made possible by incorporating a variety of study designs as different methodologies can offer distinctive insights and perspectives. To quickly determine relevance, studies were first screened using keywords and abstracts. Only those that were highly relevant moved on to the full-text evaluation stage. This review attempted to offer a thorough, targeted, and methodologically sound analysis of the research topic by utilizing these selection criteria.

Relevant information was methodically gathered to facilitate analysis and synthesis, as part of the data extraction process. The planning and execution of this process were done with great care to guarantee uniformity, precision, and thoroughness. The relevance of each study to a particular subtopic or theme within the larger research area determined how it was categorized, carefully examining full-text articles to extract pertinent details. When relevant information was discovered, it was arranged on a document into distinct sections.

The results from the studies were interpreted and summarized in an organized manner using narrative synthesis. The main conclusions of each study were condensed into a narrative that shared the main ideas and advancements made in the field. To provide a deeper understanding of the study’s implications, this step involved connecting the study’s findings to preexisting theories and models. Numerous factors, including methodological strategies, and significant results, were taken into consideration when comparing and contrasting the studies. This made it easier to find patterns, conflicts, and original contributions in the literature. 

The narrative was combined with qualitative insights to give the quantitative findings more depth and context. The narrative synthesis was structured to lead the reader through the development of research findings and their importance while maintaining a logical flow and coherence. In addition to making it easier to identify important trends and insights, this methodical approach also indicated areas that warrant further investigation, which deepened understanding of the subject matter.

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