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- Fundamentals of Enzyme Kinetics
- Enzyme Kinetics and Mechanism
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Fundamentals of Enzyme Kinetics
Enzyme assays are used extensively in hit identification and hit validation and for detailed characterization of the compound mechanism to guide lead optimization. It is important to understand the limitation of IC 50 values and to more deeply probe the relationship between the molecular structures of hits and leads and their kinetics of binding, inhibition and mechanism of action.
Combining information from enzyme kinetic studies with that derived from biophysical methods can be advantageous in assessing protein quality, generating suitable assays to identify a range of desired mechanisms and to complement detailed mechanistic characterization.
The efficient use of these methods enables the identification, prioritization and progression of truly differentiated compound series that have an enhanced probability of success in translation through to the clinic as a consequence of detailed understanding of their mechanism.
Given the therapeutic and commercial success of small-molecule enzyme inhibitors, as exemplified by kinase inhibitors in oncology, a major focus of current drug-discovery and development efforts is on enzyme targets. Understanding the course of an enzyme-catalysed reaction can help to conceptualize different types of inhibitor and to inform the design of screens to identify desired mechanisms.
Exploiting this information allows the thorough evaluation of diverse compounds, providing the knowledge required to efficiently optimize leads towards differentiated candidate drugs. This review highlights the rationale for conducting high-quality mechanistic enzymology studies and considers the added value in combining such studies with orthogonal biophysical methods.
Enzyme inhibitors and inactivators comprise roughly half of all marketed drugs 1 , 2 and have transformed human medicine. For example, angiotensin-converting enzyme ACE inhibitors 3 , including captopril 4 , 5 and lysinopril 6 , which emerged in the late s, now constitute perhaps the most important class of drugs for the treatment of hypertension.
ACE inhibitors arose from rational design based on the mechanism of action MoA of this metalloproteinase. In addition, in the s, statins, exemplified by atorvastatin 7 and lovastatin 8 , emerged as natural product-derived inhibitors of 3-hydroxyglutaryl CoA reductase and are now the most commonly used drugs to treat hypercholesterolaemia 9.
Beginning in the s, saquinivir 10 and indinavir 11 were identified as rationally designed inhibitors of the aspartic protease of HIV-1 Ref. Furthermore, in the past 20 years, ATP-competitive inhibitors of protein kinases have provided an arsenal of anticancer and anti-inflammatory drugs.
Unsurprisingly, enzymes now typically comprise one-third or more of the discrete drug targets found within the portfolios of large pharmaceutical companies. This is true even for those pharmaceutical companies that have increased their reliance on biopharmaceutical therapies. Because biopharmaceutical agents are generally confined in their utility to extracellular drug targets, intracellular enzymes still may prove to be a growth area for drug discovery.
By their very nature, enzymes are dynamic, with a single enzyme representing a number of different targets as a consequence of binding substrates, intermediates and products during the catalytic cycle.
Robertson 13 provided a useful compendium of enzymes that are the targets of marketed drugs and makes the case that unlike drug targets of other protein classes, knowledge of the chemical mechanism of an individual enzyme, including the structural characterization of its transition state 14 , 15 , 16 , 17 , the exploitation of nucleophilic, active-site residues to form covalent adducts with electrophilic inactivators 18 , 19 or the chemical conflation of two enzyme substrates into a bi-substrate analogue 20 , 21 , affords the opportunity to design de novo enzyme inhibitors based on the chemical species that emerge during catalysis.
Drugs have therefore been designed that target a number of different enzyme forms via covalent and noncovalent inhibition Fig. A single enzyme E may represent a number of different targets that may be the focus of chemical intervention to modulate activity. By their very nature, enzymes are dynamic and bind substrates S , intermediates X and products P during the catalytic cycle.
Enzyme inhibitors I may bind to these different species either covalently or non-covalently. It is possible to modulate the abundance of these different enzyme species by changing assay conditions to obtain a balance between enzyme forms to maximize the chances of finding any type of inhibitory mechanism or to increase the relative concentration of some forms in order to focus on particular mechanisms.
An early understanding of the enzyme mechanism is advantageous for the identification and subsequent characterization of hits and leads. PowerPoint slide. Understanding the role of enzymes in disease states and the implementation of strategies to modulate their activities for therapeutic benefit remains a key focus for drug discovery.
However, while enzymology is a powerful and established discipline, it often fails to receive recognition in providing scientific insight into problems experienced along the route from target identification to proof of concept in the clinic. This is particularly true at the onset of a drug discovery campaign that targets an enzyme. It is uncommon to have a detailed understanding of the kinetic mechanism the order of substrate addition and product release and the chemical mechanism of catalysis of an enzyme target before the initiation of 'hit' discovery.
Ironically, at this early stage of a programme, an X-ray structure of the enzyme target may be extant even in the absence of an understanding of its detailed mechanism of catalysis. As a result, the opportunity for the use of rational design to afford enzyme inhibitors or inactivators based on the enzyme mechanism may have been eclipsed by a medicinal chemistry effort that focuses on a limited number of chemotypes that in no way resemble the substrates of the enzyme target.
Additionally, following the knowledge gleaned from more than 20 years of 'random' high-throughput screening HTS applied to hundreds of enzyme targets, pharmaceutical companies are increasingly capable of predicting which classes of enzyme targets in their active portfolios will provide durable lead compounds from HTS campaigns and which are likely to fail to do so. For these and other reasons, mechanistic studies of enzyme targets are now enjoying a renaissance, with many pharmaceutical companies increasing their focus on using detailed, more physiologically relevant enzyme kinetic studies during the lead identification and optimization phases as another measure to address the increasingly high cost and low success rates of drug discovery within the industry.
There is renewed recognition that mechanistic enzymology is essential for establishing robust, reliable and appropriate assays for screening campaigns to identify hit compounds that are also adaptable to comprehensively characterize inhibitors more fully than traditional half-maximal inhibitory concentration IC 50 values alone.
While useful, the information inherent in IC 50 values does not indicate a change in inhibitory mechanism for example, a shift from competitive to mixed-type inhibition in a series of lead compounds and moreover, the data are poorly correlative with their pharmacodynamic action, as they give no measure of the period of time the compound 'resides' on its target The purpose of these MoA studies is to characterize the interaction of a compound with its target and to understand how natural ligands at physiological concentrations will modulate this activity.
Ideally, data from a series of hit compounds are annotated in terms of potency, selectivity versus homologous targets and their developability properties, and MoA data are delivered for all lead and drug candidates. This practice provides a full 'data dossier' for the advancement of lead compounds to clinical candidates with a well-characterized MoA, cellular efficacy and the appropriate safety and pharmacokinetic properties. This is particularly true when one considers the difficulty of registering a drug that has no known functional target Additionally, it allows the identification and prioritization of not just a single mechanism but potentially a range of diverse mechanisms, thoroughly characterized using increasingly complex assays isolated protein through to tissues , to produce a number of differentiated options to achieve the desired biological effect in vivo.
Of course, enzymology alone cannot deliver all of the required data or information needed in early drug discovery, and its combination with biophysical methods and protein structural analysis remains essential for providing an integrated pharmacological view of the thermodynamics and kinetics of enzyme catalysis and inhibition Furthermore, integrating the understanding derived from detailed mechanistic characterization in isolated enzyme assays coupled with cell-based assays and, ultimately, with metabolic pathways and systems knowledge will continue to identify enzymology as one of the most important quantitative disciplines within drug discovery.
This article will discuss the importance of conducting high-quality mechanistic enzymology studies, consider how detailed and early knowledge of an enzyme mechanism may add value throughout a drug-discovery programme and assess the value of combining such knowledge with orthogonal biophysical methods.
What is the appropriate biochemical nature of an enzyme target and its substrate? When a decision is made to pursue a discrete enzyme as a drug target, the optimal biochemical nature of that enzyme and its substrates becomes the subject of debate. Does one insist on a purely cellular environment for the enzyme and its substrates to maintain biological integrity or is one able to proceed with a purified recombinant enzyme and substrates that may not resemble its native substrates? As one example, the development of HIV-1 protease inhibitors as drugs was achieved with a recombinant decrement of the protease and with using non-protein, oligopeptide substrates resembling cognate cleavage sites in retroviral polyproteins However, as elaborated below, protein kinases provide interesting examples in which a recombinant 'biochemical' enzyme species must more closely conform to its native 'biological' entity Classical drug-discovery campaigns generally include a combination of biochemical, biophysical and cell biology assays and methodologies to identify and characterize modulators of key targets and pathways of interest.
In order to derive the most relevant insights from the various approaches with the goal of translating in vitro data to a clinical disease setting, a number of important considerations are required.
A comprehensive understanding of the molecular environment of the target of interest is crucial to enable the design of more physiologically relevant assays. Recent times have seen the pharmaceutical industry moving away from the use of isolated, usually recombinant enzyme domains, model or generic substrates and returning towards a focus on systems likely to be of higher relevance and translatability.
This has resulted in a greater number of assays employing full-length enzymes, complex partners and native substrates, requiring a more detailed analysis of the underlying enzymology. It is important to consider that in the complex physiological environment, full-length, multi-domain enzymes comprise catalytic domains that can be regulated by non-catalytic domains in a manner that controls activity, substrate specificity and potentially inhibitor structure—activity relationships SARs.
A research strategy that utilized a recombinant fragment encompassing only the catalytic domain of this membrane-associated protein kinase, rather than the native, full-length protein, would have overlooked the allosteric site on the amino-terminal pleckstrin homology domain that ultimately provided an exploitable site for inhibitor discovery While one pharmaceutical company could have decided that recombinant expression of the kinase domain of the AKT kinases would prove more expeditious and more amenable to structural characterization, Merck chose the path of producing the 'native' kinase including the pleckstrin homology domain and thereby discovered a new site for enzyme inhibition, defining a new chemical equity 26 , 27 Table 1.
In this system, the use of the isolated kinase domain would be poorly representative of the kinetic characteristics of the enzyme. Furthermore, demonstrating also the impact of protein tags, the k cat value for the hexahistidine-tagged cytoplasmic domain of hepatocyte growth factor receptor HGFR; also known as c-MET has been reported to be 0.
In an even more striking example, the addition of a amino acid juxta membrane domain to the catalytic domain of vascular endothelial growth factor receptor VEGFR increases the affinity of a small-molecule inhibitor from 1, pM to 28 pM Ref. Detailed characterization and comparison of the mechanism and key kinetic parameters between the different enzyme forms are required in order to be confident of the relevance of the isolated domain with regard to the pathophysiological state of the enzyme.
Domain architecture, post-translational modification status, activation state and artificial modifications such as affinity tags can all affect the kinetic integrity of the enzyme. Complex binding partners can also exert significant influence on the catalytic activity of the associated enzyme, a classic example being the cyclin partners of cyclin-dependent kinase CDK enzymes, where activation of the cyclin-dependent kinases is a two-step process comprising cyclin binding, followed by phosphorylation at a conserved threonine residue within the kinase activation loop Such modifications have the potential to also affect inhibitor binding.
For example, targeting protein for XKLP2 TPX2 is a physiological binding partner and positive regulator of Aurora A kinase AurA ; while the presence of TPX2 has no effect on the reaction mechanism and enzyme turnover, it does affect the binding affinity of the kinase's substrates and also the SAR of inhibitors binding to a hydrophobic pocket adjacent to the ATP binding site 35 Table 1. Profiling in the presence of the native binding partner can clearly enhance the probability of identifying molecules that are more likely to have an impact in physiological environments.
More than a single enzyme subunit may come into play. For example, the histone-lysine N -methyltransferase EZH2 can fully catalyse the methylation of histone H3 lysine 27 only in the presence of its associated partners comprising its full pentameric Polycomb repressive complex 2 Hence, an understanding of the physiologically relevant protein complex is key to ensuring that the correct form of the enzyme is represented.
Indeed, there may be specific complexes associated with health-relevant and disease-relevant states of the enzyme, the knowledge of which can help to drive selectivity. Immunoprecipitation from disease-relevant cells coupled with mass spectrometry analysis can help to identify complex partners and their associated stoichiometry The biochemical nature, or identity, of enzyme substrates is another important consideration.
There are numerous examples where binding interactions between macromolecular substrates and enzymes occur at sites distal to the active site and indeed can contribute to the overall binding energy for the formation of the initial enzyme—substrate ES encounter complex Substitution of the native substrate with, for example, in the case of a protease or kinase, a shorter peptide could affect both the kinetic mechanism of the enzyme and the ability to identify inhibitors that might prevent formation of the ES complex by binding to distal sites, particularly if specific conformational changes accompany binding of the native substrate.
For example, specific inhibitor pockets are revealed upon substrate binding in bacterial Glu-tRNA Gln amidotransferases Among protein kinases are numerous examples in which the kinetic mechanism and small-molecule affinity are directly influenced by the choice of substrate. However, in the presence of a short peptide as a phosphoacceptor, the reaction proceeds through an ordered mechanism with MgATP binding first A similar scenario is recognized for 3-phophoinositide-dependent protein kinase 1 PDK1 , in which phosphorylation of an extended peptide substrate containing a single distal recognition element reacts through a rapid-equilibrium, random-order mechanism However, phosphorylation of the native downstream substrate S6K1 protein kinase occurs through a steady-state ordered mechanism in which binding of S6K1 precludes association of MgATP Detailed understanding of the catalytic mechanism and full consideration of the various enzyme—substrate interactions can afford control over the specific downstream pathways to be inhibited in a cellular context.
For example, in the case of p38, differential substrate selectivity is able to define the particular pathways affected by small molecules, offering exquisite control and, potentially, fewer unwanted side effects 46 , 47 Table 1. For a number of years in the epigenetic field, the importance of using native nucleosome substrates in preference to short, histone N -terminal tail peptides as surrogates has been realized in order to identify small-molecule inhibitors that translate through to the cellular context 34 and target the desired activity of the enzyme.
In the case of the histone-lysine N -methyltransferase NSD2, for example, the specificity of lysine methylation is directed by the choice of a nucleosome substrate versus a histone octamer substrate in the presence of short single-stranded DNA or double-stranded DNA In order to elucidate the true structure of the transition states of the NSD2-catalysed methylation of Lys36 of histone H3, methylated, full-length nucleosomes were required as substrates to provide fidelity of the biological target in a panel of kinetic isotope effect studies This reiterates an important observation that while many enzymes can be observed to catalyse low-level activity using surrogate substrates, the physiological relevance of this needs to be verified These examples illustrate the importance of understanding the most physiologically relevant form of the enzyme to study and, indeed, target.
Moreover, if it is not always feasible to perform high-throughput hit-finding campaigns with full-length protein and native substrates, it is crucial to explore the level of compromise imbued by the use of simpler systems and define a strategy to relate any activities back to the cellular context.
A clear advantage of having detailed knowledge of the molecular environment of the enzyme in the disease-relevant setting is the ability to prioritize specific mechanisms of interest to target. For example, it may be preferable to identify inhibitors that are not competitive with the native substrate where the cellular concentration of the latter is high; an emerging focus in the field of kinase drug discovery is on the identification of inhibitors that are either non-competitive inhibitors or uncompetitive inhibitors with respect to ATP, in order to circumvent the challenge of high cellular concentrations of ATP While rare, enzyme inhibitors that operate by uncompetitive inhibition are ideal for disrupting enzyme targets within metabolic pathways because the resulting accumulation of the substrate cannot thwart inhibition as it is not competitive The rate-limiting step in the mechanism of mammalian IMPDH is the hydrolysis of a covalent thioimidate complex formed between the nascent XMP product and an active-site cysteine.
Is an understanding of the rate-limiting step of a target enzyme before the launch of a drug discovery programme essential? The rates of the majority of enzyme-catalysed reactions are limited by either chemical or product release steps.
Enzyme Kinetics and Mechanism
The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since , each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than volumes all of them still in print , the series contains much material still relevant today—truly an essential publication for researchers in all fields of life sciences. Biochemists, biophysicists, pharmacologists, molecular biologists, analytical, organic, and medicinal chemists, and graduate students in these disciplines. It should be on the shelves of all libraries in the world as a whole collection. Daniel Lee Purich has been at the forefront of biochemistry research for more than 25 years.
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Enzyme kinetics is the study of the chemical reactions that are catalysed by enzymes. Studying an enzyme's kinetics in this way can reveal the catalytic mechanism of this enzyme, its role in metabolism , how its activity is controlled, and how a drug or an agonist might inhibit the enzyme. Enzymes are usually protein molecules that manipulate other molecules—the enzymes' substrates.
Enzyme Kinetics and Mechanisms pp Cite as. In general experiments with the inhibition of initial velocity by substrate analogs is a useful method by which the best steady-state chemical model for an enzyme system can be established. The ordered binding of substrates can be distinguished from random binding, as well as various combinations of the two, in terreactant systems.
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