Asp304 of Escherichia coli acid phosphatase is involved in leaving group protonation.
Site-directed mutagenesis was used to explore the role of potential proton donors in the mechanism of the Escherichia coli acid phosphatase that is encoded by the appA gene. Asp304 appeared to be the only carboxylic acid residue that is conserved in the protein sequences of the high molecular weight acid phosphatases. The mutations Asp304Ala and Asp304Glu were introduced into appA and the corresponding proteins were overexpressed in E. coli and purified to homogeneity. Only small decreases were observed for the Km values of the substrates p-nitrophenyl phosphate, fructose 1,6-diphosphate, and tripolyphosphate. However, Vmax was greatly decreased, and the magnitude of effect depended markedly on substrate. Both mutant proteins exhibited significantly lower Vmax values with fructose 1,6-diphosphate, which possesses a much poorer leaving group than do the other two substrates. The importance of the leaving group was further tested by using a number of phenyl and alkyl phosphate derivatives as substrates. A linear correlation was observed between log Vmax and the pKa of the substrate leaving group for catalysis by the Asp304Ala mutant enzyme. These results are consistent with partition experiments using ethylene glycol as an alternate nucleophile, which indicated that for the Asp304Ala protein, the formation of a phosphoenzyme intermediate is the rate-determining step, in contrast to the situation for the wild type enzyme and the His303Ala mutant. In the latter case, the rate-limiting step of the reaction is interpreted to be the breakdown of phosphoenzyme. It is concluded that Asp304, rather than His303, is involved in protonation of the substrate leaving group.
Ostanin
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Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393.
Van Etten
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RL
D000135:Acid Phosphatase / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry*; D001483:Base Sequence; D004277:DNA, Single-Stranded; D003903:Deuterium / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007554:Isotopes; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000737:chemistry; D011522:Protons; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D007554:Isotopes; D010710:Phosphates; D011522:Protons; D001224:Aspartic Acid; D003903:Deuterium; D000135:Acid Phosphatase