A cationic cluster of amino acid residues of inorganic pyrophosphatase from Escherichia coli as a possible site of effector binding

摘要

A computer-assisted analysis of the molecule of Escherichia coli pyrophosphatase was earlier used to localize the site capable of binding free pyrophosphate (PPi) or methylenediphosphonate, a PPi analogue, and thereby activating the enzyme. A cluster of positively charged amino acid residues (Lys146, Lys148, Lys115, and Arg43) was revealed, and Lys115Ala, Lys 148Gln, and Arg43Gln mutant pyrophosphatases (PPases) were obtained. It was shown that the kinetics of hydrolysis of the magnesium pyrophosphate (MgPPi) substrate by these mutant variants does not obey the Michaelis-Menten equation, which is expressed in two slopes in the double-reciprocal plots of the enzyme reaction rate vs. substrate concentration. The two regions on the curves correspond to the ranges of high and low MgPPi concentrations. This suggests that, in all mutant variants of the enzyme, the binding of PPi at the effector site weakens, whereas the affinity of MgPPi for the active site remains practically unchanged. Other properties of the enzymes, such as their oligomeric states, resistance to thermal denaturation, and resistance to the denaturing agent guanidine hydrochloride, were thoroughly studied. The constants of binding of Mg2+ to mutant enzymes in the absence of substrate and to enzyme-substrate complexes were determined. The introduction of amino acid substitutions was shown to stabilize the protein globule.