Activity and structure of the active-site mutants R386Y and R386F of Escherichia coli aspartate aminotransferase. Academic Article uri icon

Overview

abstract

  • Arginine-386, the active-site residue of Escherichia coli aspartate aminotransferase (EC 2.6.1.1) that binds the substrate alpha-carboxylate, was replaced with tyrosine and phenylalanine by site-directed mutagenesis. This experiment was undertaken to elucidate the roles of particular enzyme-substrate interactions in triggering the substrate-induced conformational change in the enzyme. The activity and crystal structure of the resulting mutants were examined. The apparent second-order rate constants of both of these mutants are reduced by more than 5 orders of magnitude as compared to that of wild-type enzyme, though R386Y is slightly more active than R386F. The 2.5-A resolution structure of R386F in its native state was determined by using difference Fourier methods. The overall structure is very similar to that of the wild-type enzyme in the open conformation. The position of the Phe-386 side chain, however, appears to shift with respect to that of Arg-386 in the wild-type enzyme and to form new contacts with neighboring residues.

publication date

  • February 19, 1991

Research

keywords

  • Aspartate Aminotransferases
  • Escherichia coli
  • Mutagenesis, Site-Directed

Identity

Scopus Document Identifier

  • 0025809948

Digital Object Identifier (DOI)

  • 10.1021/bi00221a035

PubMed ID

  • 1993208

Additional Document Info

volume

  • 30

issue

  • 7