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

J Inorg Biochem. 1988 Jan;32(1):1-6. doi: 10.1016/0162-0134(88)80010-2.

Abstract

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.

MeSH terms

  • Carbon Isotopes
  • Carboxypeptidases / metabolism*
  • Carboxypeptidases A
  • Cobalt / metabolism*
  • Kinetics
  • Magnetic Resonance Spectroscopy / methods
  • Phenylalanine / metabolism*
  • Protein Binding
  • Stereoisomerism

Substances

  • Carbon Isotopes
  • Cobalt
  • Phenylalanine
  • Carboxypeptidases
  • Carboxypeptidases A