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The mode of binding to thermolysin of the unsubstituted phosphoramidate
inhibitor N-phosphoryl-L-leucinamide (P-Leu-NH2) has been determined
crystallographically and refined at high resolution (R = 17.9% to 0.16-nm
resolution). The mode of binding of the naturally occurring thermolysin
inhibitor phosphoramidon reported previously [Weaver, L. H., Kester, W. R. and
Matthews, B. W. (1977) J. Mol. Biol. 114, 119-132] has also been confirmed by
crystallographic refinement (R = 17.4% to 0.23-nm resolution). Phosphoramidon
binds to the enzyme with a single oxygen of the phosphoramidate moiety as a zinc
ligand. Together with three ligands to the metal from the protein the resultant
complex has approximately tetrahedral geometry. However, in the case of
P-Leu-NH2, two of the phosphoramidate oxygens interact with the zinc to form a
complex that tends towards pentacoordinate. In this respect, P-Leu-NH2 appears
to be a better transition-state analog than is phosphoramidon. In addition, the
phosphorus-nitrogen bond length in P-Leu-NH2 is 0.18 nm, suggesting that the
nitrogen is protonated whereas the same bond in phosphoramidon is much shorter
(0.15 nm) suggesting that the nitrogen does not carry a charge. In
phosphoramidon the distance from the phosphoramide nitrogen to Glu-143 is 0.39
nm whereas in P-Leu-NH2 this distance decreases to 0.34 nm. Taken together,
these observations provide additional evidence in support of the participation
of pentacoordinate intermediates in the mechanism of action of thermolysin
[Holmes, M. A. and Matthews, B. W. (1981) Biochemistry 20, 6912-6920] and the
role of Glu-143 in first promoting the attack of a water molecule on the
carbonyl carbon of the scissile bond and subsequently acting as a 'proton
shuttle' to transfer the proton to the leaving nitrogen [Monzingo, A. F. and
Matthews, B. W. (1984) Biochemistry 23, 5724-5729; Hangauer, D. G., Monzingo, A.
F. and Matthews, B. W. (1984) Biochemistry 23, 5730-5741].
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