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The functional role of tyrosine-63 in the catalytic action of human lysozyme (EC
3.2.1.17) has been probed by site-directed mutagenesis. In order to identify the
role of Tyr63 in the interaction with substrate, both the three-dimensional
structures and the enzymatic functions of the mutants, in which Tyr63 was
converted to phenylalanine, tryptophan, leucine, or alanine, have been
characterized in comparison with those of the wild-type enzyme. X-ray
crystallographical analysis of the mutant enzyme at not less than 1.77-A
resolution indicated no remarkable change in tertiary structure except the side
chain of 63rd residue. The conversion of Tyr63 to Phe or Trp did not change the
enzymatic properties against the noncharged substrate (or substrate analogs)
largely, while the conversion to Leu or Ala markedly reduced the catalytic
activity to a few percent of wild-type enzyme. Kinetic analysis using
p-nitrophenyl penta-N-acetyl-beta-(1----4)-chitopentaoside (PNP-(GlcNAc)5) as a
substrate revealed that the reduction of activity should mainly be attributed to
the reduction of affinity between enzyme and substrate. The apparent
contribution of the phenolic hydroxyl group and the phenol group in the side
chain of Tyr63 was estimated to 0.4 +/- 0.4 and 2.5 +/- 0.8 kcal mol-1,
respectively. The result suggested that the direct contact between the planar
side-chain group of Tyr63 and the sugar residue at subsite B is a major
determinant of binding specificity toward a electrostatically neutral substrate
in the catalytic action of human lysozyme.
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