PH-DEPENDENT PROPERTIES OF COBALT(II) CARBOXYPEPTIDASE-A INHIBITOR COMPLEXES

H-1 NMR spectroscopy of the isotropically shifted signals in cobalt carboxypeptidase, CoCPD, permits a direct and selective detection of protons belonging to the residues liganded to the metal. The chemical shift of these protons in the free enzyme and enzyme inhibitor complexes with changing pH monitors the state of ionization of the ligands directly and of other residues in the active center indirectly. The H-1 NMR spectrum of CoCPD at pH 6 shows three well-resolved isotropically shifted signals in the downfield region at 62 (a), 52 (c), and 45 (d) ppm which have been assigned to the NH proton of His-69 and to the C-4 H's of His-69 and His-196, respectively. Titration of signal a with pH is characterized by a pK(a) of 8.8 which is identical to that seen in prior electronic absorption and kinetic studies. The fact that the signal reflecting the NH of His-69 is still observed at pH 10 and no major shifts occur for the signals reflecting the C-4 H's indicates the alkaline pK(a) in carboxypeptidase A catalysis, pK(EH), cannot be ascribed to ionization of the histidyl NH of either His-69 or His-196. Binding of L-Phe shifts this pK(a) to 7.7 while not greatly perturbing the downfield H-1 NMR signals that reflect the ligation shell of the cobalt coordination sphere. These results indicate the pK(a) of 8.8 in CoCPD and the pK(a) of 7.7 in the CoCPD.L-Phe adduct reflect ionization of the same group. In conjunction with previous kinetic studies Of L-Phe inhibition, it can now be ascertained that the protonated alpha-amino form of L-Phe (IH) binds 50-fold tighter to the ionized form of the enzyme (E). L-Phe binding as its protonated alpha-amino group likely disrupts the Glu-270 carboxylate-metal water interaction by forming a salt link between the Glu-270 carboxylate and the a-amino group. H-1 NMR shows N3- binds to the protonated adduct, EHIH, while a second D-Phe binds to the ionized adduct EHI. The roles of the ionization of the metal-coordinated water and Tyr-248 in these processes are discussed.