13C NMR Studies of D- and L-Phenylalanine Binding to Cobalt(II) Carboxypeptidase A Claudio Luchinat, Roberto Monnanni, Stefano Roelens, Bert L. Vallee, and David S. Auld CL., RM. Department of Chemistry, University of Florence, Italy.--SR. Center for the Study of the Chemistry and Structure of Heterocyclic Compounds, C.N.R., Florence, Italy.--BV., DA. Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School ABSTRACT '3C NMR 7"1 and T2 measurements have been performed on cobalt(II) substituted carboxypeptidase A in the presence of carboxylate-~3C-enriched L- and D-phenylalanine. Upon binding to the cobalt enzyme, the longitudinal and transverse relaxation rates Tip- l and T2p- l of these inhibitors are enhanced significandy compared to the zinc enzyme, allowing both determination of an affinity constant for inhibitor binding, K, and calculation of the metal-t3C carboxylate distances. The L-and D- Phe concentration dependence of T2p-~ yields affinity constants of 290 + 60M-1 and 670 + 90M-1. The distance measurements calculated for Co-13C from Tip- 1are 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. ABBREVIATIONS CPD-A, carboxypeptidase-A; NMR, nuclear magnetic resonance; FID, free induction decay; MCD, magnetic circular dichroism; Hepes, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid; pH*, apparent pH measured with glass electrode. INTRODUCTION Binding of anionic ligands to the active site metal ion of carboxypeptidase A (CPD-A) in solution has recently been discovered to be greatly facilitated by the presence of C- Address reprint requests to Prof. Claudio Luchinat, Department of Chemistry, University of Florence, Via Gino Capponi 7, 50121 Florence, Italy. Journal oflnorganic Biochemistry 32, 1-6 (1988) 1 © 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, IVY 10017 0162-0134/88/$3.50