Specificity of Non-Michaelis−Menten Enzymes: Necessary Information for Analyzing Metabolic Pathways

Abstract

The specificity of an enzyme obeying the Michaelis-Menten equation is normally measured by comparing the k cat /K m for different substrates, but this is inappropriate for enzymes with a Hill coefficient h different from 1. The obvious alternative of generalizing K m in the expression as K 0.5 , the substrate concentration for half-saturation, is better, but it is not entirely satisfactory either, and here we show that k cat /K 0.5 h gives satisfactory results for analyzing the kinetic behavior of metabolic pathways. The importance of using k cat /K 0.5 h increases with the value of h, but even when h is small, it makes an appreciable difference, as illustrated for the mammalian hexokinases. Reinterpretation of data for the specificity of these enzymes in terms of the proposed definition indicates that hexokinase D, often believed highly specific for glucose, and accordingly called "glucokinase", actually has the lowest preference for glucose over fructose of the four isoenzymes found in mammals.

Changing the Selectivity of p300 by Acetyl-CoA Modulation of Histone Acetylation

“…Specificity is the ability of an enzyme to target a specific substrate, or in the case of p300, to target a specific lysine residue. This parameter is usually reported as k cat /K m nH 26 . Selectivity is the ability of p300 to target one specific residue relative to another residue.…”

A quantitative multiplexed mass spectrometry assay for studying the kinetic of residue-specific histone acetylation

“…Specificity of a KAT for each lysine is defined by the specificity constant, k cat /K m(app) [39] (or k cat /K 1/2 nH if the sigmoidal curve is present [40]), which is a quantitative parameter to indicate the capability of a KAT to catalyze a site/substrate. In addition, we can compare the differences of these specificity constants between different lysines to understand how a KAT selectively acetylates one site over the other.…”

The UDP-glucose pyrophosphorylase from Giardia lamblia is redox regulated and exhibits promiscuity to use galactose-1-phosphate

“…Enzyme kinetics for TDP-Glc synthesis showed lower value of V max (30-fold) as well as a higher S 0.5 for substrates (6-fold for TTP and 3-fold for Glc-1P); with a slight increase in the n value for both substrates when compared with the production of UDP-Glc (see Table 1). Then, the catalytic efficiency (defined as V max /(S 0.5 ) n , analogous to V max /K m for hyperbolic kinetics [25]) of GlaUDP-Glc PPase for using UTP was about two orders of magnitude higher than for TTP.…”

“…The experimental data were plotted as enzyme activity (U/mg) versus substrate concentration (mM), and kinetic constants were determined by fitting the data to the modified Hill equation: v o = V max [S] n / (S 0.5 n + [S] n ), as described elsewhere [24], using the LevenbergMarquardt nonlinear least-squares algorithm provided by the computer program Origin™ 8.0. Hill plots were used to calculate the Hill coefficient (n), measuring the interaction degree (cooperativity) between kinetically different binding sites per mole of enzyme [25,26]; the maximal velocity (V max ); and the kinetic constants that correspond to the substrate concentrations giving 50% of the maximal velocity (S 0.5 ).…”