13C NMR studies of D- and L-phenylalanine binding to cobalt(II) carboxypeptidase A.
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.
Luchinat
C
C
Department of Chemistry, University of Florence, Italy.
Monnanni
R
R
Roelens
S
S
Vallee
B L
BL
Auld
D S
DS
D002247:Carbon Isotopes; D002268:Carboxypeptidases / Q000378:metabolism*; D043422:Carboxypeptidases A; D003035:Cobalt / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010649:Phenylalanine / Q000378:metabolism*; D011485:Protein Binding; D013237:Stereoisomerism
D002247:Carbon Isotopes; D003035:Cobalt; D010649:Phenylalanine; D002268:Carboxypeptidases; D043422:Carboxypeptidases A