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13C NMR studies of d- and l-phenylalanine binding to cobalt(II) carboxypeptidase A

1988; Elsevier BV; Volume: 32; Issue: 1 Linguagem: Inglês

10.1016/0162-0134(88)80010-2

1873-3344

Claudio Luchinat, Roberto Monnanni, Stefano Roelens, Bert L. Vallée, David S. Auld,

Metal-Catalyzed Oxygenation Mechanisms

13C NMR T1 and T2 measurements have been performed on cobalt(II) substituted Carboxypeptidase A in the presence of carboxylate-13C-enriched l- and d-phenylalanine. Upon binding to the cobalt enzyme, the longitudinal and transverse relaxation rates T1p−1 and T2p−1 of these inhibitors are enhanced significantly compared to the zinc enzyme, allowing both determination of an affinity constant for inhibitor binding, K, and calculation of the metal-13C carboxylate distances. The l-and d- Phe concentration dependence of T2p−1 yields affinity constants of 290 ± 60M−1 and 670 ± 90M−1. The distance measurements calculated for Co-13C from T1p−1 are 0.39 ± 0.04 and 0.42 ± 0.04 nm for l-Phe and d-Phe. Both values are too great for direct coordination of their carboxylate groups to the metal atom. Upon formation of their respective ternary enzyme · Phe · N3− complexes, the distances are essentially unaltered. In conjunction with electronic absorption studies on these complexes it can be concluded that N3−, but not the amino acid carboxylate, is bound to the metal.