Regulatory mutants of Escherichia coli glycerol kinase: initial-velocity and fructose 1,6-bisphosphate binding studies

Abstract

This work constitutes the first detailed report of structure perturbation studies aimed at understanding mechanism of regulation and the catalytic activity of Escherichia coli glycerol kinase. These studies consist of purification and investigation of the catalytic and regulatory properties of three mutant glycerol kinases, A65T (alanine 65 is replaced by threonine), D72N (aspartate 72 is replaced by asparagine), and G304S (glycine is replaced by serine). These mutations were identified by screening after mutagenesis, i.e., cells containing a glycerol kinase that is no longer sensitive to regulation by fructose 1,6-bisphosphate (FBP). AU three mutants behave the same as the wild type enzyme during the purification. The mutations show two classes of effects on the catalytic properties, depending on the region in which the mutation occurs. The mutations A65T and D72N have little effects on the catalytic properties of glycerol kinase. These two mutations have no significant effects on the vmax, Michaelis constants, substrate dissociation constants, or the apparent substrate activation by MgATP at higher glycerol concentrations. These two enzymes are inactivated by MgATP at lower glycerol concentrations. Mutation G304S strongly affects most of the catalytic properties, which is consistent with its location near the active site. It increases both Michaelis constants greatly, and it appears to eliminate substrate activation by ATP. Apparent substrate inhibition by MGATP, rather than substrate activation by ATP, is observed at all glycerol concentrations for G304S. The gel permeation chromatography experiments show that the molecular sizes of both A65T and D72N glycerol Kinase correspond to the dimer in the absence of fructose 1,6-bisphosphate (FBP), while the molecular size of the wild type enzyme corresponds that of the tetramer. Addition of FBP shifts the molecular size of the D72N enzyme to that of the tetramer, while the size of the A65T enzyme increases but not completely to that of the tetramer. Neither the molecular size of the G304S glycerol kinase nor the catalytic activity is affected by FBP. The activities of A65T and D72N are inhibited by IIIglc; G304S appears to eliminate the regulation by IIIglc.