[A cationic cluster of amino acid residues of inorganic pyrophosphatase from Escherichia coli as a possible site of effector binding].
A computer-assisted analysis of the molecule of Escherichia coli pyrophosphatase was earlier used to localize the site capable of binding free pyrophosphate or methylene diphosphonate, 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, Lys148Gln, 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 plot 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 becomes worse, whereas the affinity of MgPPi for the active site remains practically unchanged. Other properties of the enzymes, such as its oligomeric state, 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 the substrate and to enzyme-substrate complexes were determined. The introduction of amino acid substitutions was shown to stabilize the protein globule. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.
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D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D011756:Diphosphates / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000235:genetics; D008274:Magnesium / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics
D011756:Diphosphates; D029968:Escherichia coli Proteins; D043564:Inorganic Pyrophosphatase; D008274:Magnesium