Effects on general acid catalysis from mutations of the invariant tryptophan and arginine residues in the protein tyrosine phosphatase from Yersinia.
General acid catalysis in protein tyrosine phosphatases (PTPases) is accomplished by a conserved Asp residue, which is brought into position for catalysis by movement of a flexible loop that occurs upon binding of substrate. With the PTPase from Yersinia, we have examined the effect on general acid catalysis caused by mutations to two conserved residues that are integral to this conformation change. Residue Trp354 is at a hinge of the loop, and Arg409 forms hydrogen bonding and ionic interactions with the phosphoryl group of substrates. Trp354 was mutated to Phe and to Ala, and residue Arg409 was mutated to Lys and to Ala. The four mutant enzymes were studied using steady state kinetics and heavy-atom isotope effects with the substrate p-nitrophenyl phosphate. The data indicate that mutation of the hinge residue Trp354 to Ala completely disables general acid catalysis. In the Phe mutant, general acid catalysis is partially effective, but the proton is only partially transferred in the transition state, in contrast to the native enzyme where proton transfer to the leaving group is virtually complete. Mutation of Arg409 to Lys has a minimal effect on the K(m), while this parameter is increased 30-fold in the Ala mutant. The k(cat) values for R409K and for R409A are about 4 orders of magnitude lower than that for the native enzyme. General acid catalysis is rendered inoperative by the Lys mutation, but partial proton transfer during catalysis still occurs in the Ala mutant. Structural explanations for the differential effects of these mutations on movement of the flexible loop that enables general acid catalysis are presented.
Hoff
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RH
Department of Chemistry, Utah State University, Logan 84322-0300, USA.
Hengge
A C
AC
Wu
L
L
Keng
Y F
YF
Zhang
Z Y
ZY
D000143:Acids / Q000737:chemistry; D001120:Arginine / Q000737:chemistry / Q000235:genetics*; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009587:Nitrogen Isotopes; D009596:Nitrophenols / Q000737:chemistry; D010103:Oxygen Isotopes; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics*; D011522:Protons; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics*; D015007:Yersinia / Q000201:enzymology* / Q000235:genetics
D000143:Acids; D009587:Nitrogen Isotopes; D009596:Nitrophenols; D010103:Oxygen Isotopes; D011522:Protons; D014364:Tryptophan; D001120:Arginine; D017027:Protein Tyrosine Phosphatases; C024836:4-nitrophenol