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The mode of binding of the irreversible thermolysin inhibitor
ClCH2CO-DL-(N-OH)Leu-OCH3 [Rasnick, D., & Powers, J.C. (1978) Biochemistry
17, 4363-4369] has been determined by X-ray crystallography at a resolution of
2.3 A and the structure of the covalent complex refined to give a
crystallographic residual of 17.0%. This is the first such structural study of
an active-site-directed covalent complex of a zinc protease. As anticipated by
Rasnick and Powers, the inhibitor alkylates Glu-143 in the thermolysin active
site, and the hydroxamic acid moiety coordinates the zinc ion. The formation of
the covalent complex is associated with a significant shift in a segment of the
polypeptide backbone in the vicinity of the active site. This conformational
adjustment appears to be necessary to relieve steric hindrance which would
otherwise prevent alkylation of Glu-143. It is suggested that this steric
hindrance, which occurs for thermolysin but would not be expected for
carboxypeptidase A, accounts for the previously inexplicable difference in
reactivity of these two metalloproteases toward N-haloacetyl amino acids. The
relevance of this steric hindrance to the mechanism of catalysis is discussed.
In agreement with previous results [Kester, W. R., & Matthews, B. W. (1977)
Biochemistry 16, 2506-2516], it appears that steric hindrance prevents the
direct attack of Glu-143 on the carbonyl carbon of an extended substrate,
therefore ruling out the anhydride pathway in thermolysin-catalyzed hydrolysis
of polypeptide substrates and their ester analogues.
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