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. 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 v_max, substrate concentration [S], and Michaelis constant K_m, which is experimentally determined as the concentration of substrate required to attain 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 (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 k_cat = v_max /[E_t].

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.