Enzyme Mechanisms

What are the properties of enzymes?

● Enzymes are biological catalysts that alter reaction rates without changing the overall delta G or Keq and are not consumed by the reaction. Substrates bind with high specificity to the enzyme active site, which is a cleft or pocket in the protein structure where the catalyzed reaction takes place.

● Stabilizing the transition state is one of the key mechanisms of enzyme catalysis and is the molecular basis for tight binding of transition state analogs, which often function as enzyme inhibitors.

● The activation energy (delta G) is the difference between the ground state energy of the reactant and the transition state energy. Enzymes lower delta G by providing a favorable physical and chemical environment in the active site to promote catalysis.

● Cofactors provide additional reactive groups to the enzyme active site that complement the limited chemistry of amino acid side chains. Some cofactors are inorganic ions. Organic cofactors are called coenzymes, many of which are derived from vitamins.

How do enzymes function as biological catalysts in cells?

● Enzymes lower the activation energy ( delta G) of a reaction in three different ways: (1) by stabilizing the transition state, which lowers the activation barrier; (2) by providing an alternative path for product formation through reaction intermediates; and (3) by orienting the substrates appropriately for the reaction to occur.

● Functional groups in the active site mediate three main types of catalytic reaction mechanisms: (1) acid–base catalysis, (2) covalent catalysis, and (3) metal-ion catalysis.

● Enzymes perform three main types of work in the cell: (1) coenzyme-dependent redox reactions associated with energy conversion; (2) metabolite transformation reactions to interconvert metabolites in anabolic and catabolic pathways; and (3) reversible covalent modification reactions to control cell signaling processes and enzyme activity.

● Enzyme-catalyzed redox reactions in the cell often require coenzymes such as NAD+/NADH, NADP+/NADPH, FAD/FADH2, or FMN/FMNH2. These redox reactions involve the transfer of a pair of electrons or a single electron through a radical intermediate.

● Metabolite transformations in metabolic pathways most often involve isomerization reactions, condensation reactions, or hydrolysis or dehydration reactions.

● One of the most common types of reversible covalent modification reactions in cells is the addition and removal of a phosphoryl group in biomolecules. Enzymes that attach phosphoryl groups are called kinases, and enzymes that remove phosphoryl groups are called phosphatases.

What are some examples of enzyme reaction mechanisms?

● Acid–base and covalent catalysis are common in enzyme mechanisms. Chymotrypsin is an example of an enzyme that uses both acid–base and covalent catalysis during the cleavage of a peptide bond. In addition, a tetrahedral intermediate is formed that resembles the transition state conformation.

● A key feature of serine proteases is the presence in the enzyme active site of three amino acids called the catalytic triad, which consists of a catalytic serine residue plus histidine and aspartate residues that function to convert the serine residue into a highly reactive nucleophile.

● Metal ions can play multiple roles in catalysis. As an example, enolase catalyzes the dehydration of 2-phosphoglycerate to form phosphoenolpyruvate in a twostep mechanism that involves both acid–base and metal-ion catalysis. The metal ions in this reaction are necessary for ionic interactions with the substrate and intermediate.

How are enzyme kinetic parameters used to characterize enzyme function?

● Enzyme kinetics is the quantitative analysis of reaction rate data obtained with purified enzymes and defined laboratory conditions. We can use enzyme kinetic parameters to compare the catalytic efficiency of related enzymes under a variety of conditions.

● Michaelis–Menten enzyme kinetics provides a way to analyze a first-order reaction under steady-state conditions in order to relate the initial velocity v0 to the maximum velocity vmax, substrate concentration [S], and Michaelis constant Km, which is experimentally determined as the concentration of substrate required to attain vmax.

● The values of vmax and Km for an enzyme reaction are obtained from experiments in which data are collected under steady-state conditions when the concentration of the enzyme–substrate complex [ES] is minimally changing (substrate binding to enzyme is rate limiting). Product formation is measured over time for several different initial substrate concentrations.

● Plotting experimental rate data as initial velocity v0 (which is the slope of the line [P]/time) versus initial [S] produces a Michaelis–Menten plot that is hyperbolic if the enzyme reaction follows simple Michaelis–Menten kinetics.

● The calculated efficiency of an enzyme is called the turnover number kcat, which is a measure of how well an enzyme functions in the reaction. Turnover number is defined as kcat = vmax /[Et].

What are the mechanisms by which enzyme activity is regulated?

● Enzyme regulation is mediated by both enzyme bioavailability (amount of enzyme in the cell and where it is located) and catalytic efficiency (how well an enzyme works).

● Catalytic efficiency of an enzyme is regulated by reversible and irreversible inhibition, allosteric control, covalent modification, and proteolytic processing. Irreversible inhibition occurs when the inhibitor forms a covalent bond (or very strong noncovalent interaction) with the enzyme.

● The three types of reversible inhibition are (1) competitive inhibition, (2) uncompetitive inhibition, and (3) mixed inhibition, which can be distinguished from each other using enzyme kinetic data.

● The three most common ways that enzymes are regulated by covalent modification are the addition and removal of (1) phosphoryl groups, (2) methyl or acetyl groups, and (3) NMP groups, primarily adenylyl and uridylyl groups.

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