Probing essential water in yeast pyrophosphatase by directed mutagenesis and fluoride inhibition measurements.
The pattern of yeast pyrophosphatase (Y-PPase) inhibition by fluoride suggests that it replaces active site Mg(2+)-bound nucleophilic water, for which two different locations were proposed previously. To localize the bound fluoride, we investigate here the effects of mutating Tyr(93) and five dicarboxylic amino acid residues forming two metal binding sites in Y-PPase on its inhibition by fluoride and its five catalytic functions (steady-state PP(i) hydrolysis and synthesis, formation of enzyme-bound PP(i) at equilibrium, phosphate-water oxygen exchange, and Mg(2+) binding). D117E substitution had the largest effect on fluoride binding and made the P-O bond cleavage step rate-limiting in the catalytic cycle, consistent with the mechanism in which the nucleophile is coordinated by two metal ions and Asp(117). The effects of the mutations on PP(i) hydrolysis (as characterized by the catalytic constant and the net rate constant for P-O bond cleavage) were in general larger than on PP(i) synthesis (as characterized by the net rate constant for PP(i) release from active site). The effects of fluoride on the Y-PPase variants confirmed that PPase catalysis involves two enzyme.PP(i) intermediates, which bind fluoride with greatly different rates (Baykov, A. A., Fabrichniy, I. P., Pohjanjoki, P., Zyryanov, A. B., and Lahti, R. (2000) Biochemistry 39, 11939-11947). A mechanism for the structural changes underlying the interconversion of the enzyme.PP(i) intermediates is proposed.
Pohjanjoki
P
P
Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland.
Fabrichniy
I P
IP
Kasho
V N
VN
Cooperman
B S
BS
Goldman
A
A
Baykov
A A
AA
Lahti
R
R
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000187:drug effects; D011756:Diphosphates / Q000378:metabolism; D005459:Fluorides / Q000378:metabolism* / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014867:Water / Q000378:metabolism*; D015003:Yeasts / Q000201:enzymology*
D011756:Diphosphates; D014867:Water; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium; D005459:Fluorides