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Eur J Biochem. 1999 Feb;259(3):809-14.

Probing the function of the conserved tryptophan in the flexible loop of the Yersinia protein-tyrosine phosphatase.

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1
Department of Molecular Pharmacology, Albert Einstein College of Medicine, New York, USA.

Abstract

The involvement of the strictly conserved Trp354 residue in the catalysis of the Yersinia protein tyrosine phosphatase (PTPase) has been investigated by site-directed mutagenesis and kinetic studies. Crystallographic structural data have revealed that Trp354 interacts with the active site Arg409 and is located at one of the hinge positions of the flexible surface loop (WpD loop) which also harbors the general acid/base (Asp356) essential for catalysis [Schubert, H. L., Fauman, E. B., Stuckey, J. A., Dixon, J. E. & Saper, M. A. (1995) Protein Sci. 4, 1904-1913]. Two mutants were constructed and expressed that contained the Trp354-->Phe and Trp354-->Ala substitutions. The K(m) of the W354F and W354A mutants were not significantly different from that of the wild-type. However, a major decrease in the affinity for oxyanions was observed for the mutants, which is consistent with Trp354 playing a role in aligning Arg409 for oxyanion binding. In addition replacement of Trp354 with Phe or Ala caused a decrease in kcat of 200-fold and 480-fold, respectively, and impaired the ability of the mutant enzymes to stabilize the negative charge in the leaving group at the transition state. In fact, the W354F and W354A mutants exhibited catalytic efficiency and leaving group dependency similar to those observed for the general acid-deficient PTPase D356N. These results indicate that Trp354 is an important residue that keeps the WpD loop in a catalytically competent conformation and positions the general acid/base Asp356 in the correct orientation for proton transfer.

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