Enzyme: Kinetics

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. Enzyme kinetic parameters are used to compare the catalytic efficiency of related enzymes under a variety of conditions.

• The velocity v of an enzyme reaction is the product of the rate constant k and substrate concentration [S], where k refers to the rate at which S → P under standard 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 v_max, substrate concentration [S], and Michaelis constant K_m. K_m is experimentally determined as the concentration of substrate required to attain ½ v_max.

• The values of v_max and K_m 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. 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.

• A Lineweaver–Burk plot is a double reciprocal plot of enzyme kinetic data that transforms the Michaelis–Menten plot into a linear plot that can be used to estimate values for v_max and K_m.

• The calculated efficiency of an enzyme is called the turnover number k_cat, which is a measure of how well an enzyme functions in the reaction. Turnover number is defined as k_cat = v_max/[E_t].

• Enzyme reaction rates are affected by pH and temperature, which reflect physical and chemical changes in the active site under suboptimal conditions.

What are the different types of reversible and irreversible enzyme inhibitors?

• Enzymes are subject to both reversible inhibition, due to the noncovalent binding of small biomolecules or proteins to the enzyme subunit, and irreversible inhibition, in which the inhibitory molecule forms a covalent bond with catalytic groups in the enzyme active site.

• Malonate is a reversible inhibitor of the enzyme succinate dehydrogenase that competes with succinate, the normal substrate of the enzyme, for active site binding. When malonate levels are high relative to succinate, then the succinate dehydrogenase reaction is inhibited.

• There are three types of reversible inhibitors: competitive inhibitors, uncompetitive inhibitors, and mixed inhibitors, which can be distinguished from each other by using enzyme kinetic data.

• Competitive inhibitors interfere with substrate binding at the active site by fully or partially obstructing the substrate from binding. Competitive inhibitors have no effect on v_max but increase the apparent K_m.

• Uncompetitive inhibitors bind to enzyme–substrate complexes, most often outside of the enzyme active site. Uncompetitive inhibitors do not bind to the free enzyme and decrease both K_m and v_max.

• Mixed inhibitors function as competitive and uncompetitive inhibitors by binding to both the free enzyme (E) and to the enzyme–substrate complex (ES). Mixed inhibitors decrease v_max and increase or decrease the K_m depending on the relative values of the dissociation constants K_I and K_I′ (that is, K_I′ > K_I or K_I > K_I′).

• Noncompetitive inhibitors are a special case of mixed inhibition in which the dissociation constants K_I and K_I′ for the free enzyme and for the enzyme–substrate complex are equal (K_I = K_I′). Noncompetitive inhibitors decrease v_max with no change in the K_m value.

• Irreversible inhibitors form a covalent bond (or very strong noncovalent interaction) with the enzyme to block its activity. Irreversible inhibitors may contain a highly reactive chemical group that forms a covalent bond with the protein as a result of specific binding to the enzyme.

• Suicide inhibitors are a type of irreversible inhibitor in which the inhibitor must first be chemically modified by the catalytic activity of the enzyme before it is able to form the inactivating covalent bond within the protein.

What are the mechanisms by which enzyme activity is regulated?

• Enzyme regulation is mediated by both enzyme bioavailability (the 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.

• The enzyme aspartate transcarbamoylase (ATCase) contains regulatory subunits and catalytic subunits. ATCase is allosterically inhibited by CTP and allosterically stimulated by ATP. CTP and ATP are heterotropic allosteric regulators of ATCase activity because their binding to the regulatory subunits affects substrate binding to the catalytic subunits.

• 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.

• Glycogen phosphorylase is an example of an enzyme that is regulated by covalent modification; the phosphorylated form is in the active R-state conformation, whereas the unphosphorylated form is in the inactive T-state conformation.

• The adenylylated form of the enzyme glutamine synthetase is in the inactive T-state conformation and the deadenylated form is in the active R-state conformation. Glutamine synthetase adenylyltransferase regulates glutamine synthetase through its adenylylating and deadenylylating activity.

• Glutamine synthetase adenylyltransferase is itself regulated by uridylylation; the uridylylated form of glutamine synthetase adenylyltransferase has deadenylylating activity, whereas the deuridylylated form has adenylating activity.

• Zymogens are inactive proenzymes that are irreversibly processed by proteolysis to generate the active form of the enzyme. Two examples are pepsinogen and chymotrypsinogen, both of which are digestive enzymes that are tightly controlled to prevent aberrant proteolysis.

Everyday example: CO-8: Statins Inhibit Cholesterol Synthesis