Azide and chloride binding to carboxypeptidase A in the presence of L-phenylalanine

Abstract

The interaction of chloride with native and cobalt (Co)-substituted carboxypeptidase-A (CPD) has been investigated by 35Cl nuclear magnetic resonance (NMR) spectroscopy in the presence and absence of L-Phe. The affinity constants of azide and chloride toward the Co(II)CPD·L-Phe complex have been measured by electronic spectroscopy. The correlation times determining T 1 and T 2 for the 35Cl nuclei are related to movements inside the cavity. In the presence of L-Phe, the anions bind to the metal with a relatively high affinity at pH values below 6. Anion binding to the Co enzyme can be analyzed in terms of the three protonation state model for the enzyme (EH 2 α EH α E). In the presence of L-Phe, Cl − binds to EH 2 but not to EH or E while N 3 − binds ∼ 34 times tighter to EH 2 than EH, while binding to E is undetectable. The pH-binding constant profile for N 3 − binding to the Co(II)CPD·L-Phe complex is characterized by pK a values of 5.3 and 7.6. The lower value corresponds to the pK a of the group that characterizes the pH- k cat profile for Co(II)CPD while the higher pK a likely reflects the group that characterizes the pH-pK m profile in the alkaline pH region. This group has a pK a of 8.9 in the Co and native enzymes but is shifted to 7.6 in the presence of L-Phe. The present results in conjunction with prior spectral and kinetic studies suggest azide preferentially binds to the enzyme when L-Phe has its amino group protonated. The highly synergistic binding between L-Phe to the active site and azide to the metal may be viewed as caused by replacement of the Glu-270 metal-water hydrogen-bonding network by an electrostatic interaction between ionized Glu-270 and the protonated amino group of L-Phe. Thus, the formation of this E·I species allows ready displacement of the metal bound water by azide.