Investigation of intermediates and transition states in the catalytic mechanisms of active site substituted cobalt(II), nickel(II), zinc(II), and cadmium(II) horse liver alcohol dehydrogenase.

Abstract

Active site substituted Co(II)-, Ni(II)-, and Cd(II)-horse liver alcohol dehydrogenase derivatives are compared to the native Zn(II)-enzyme with respect to the kinetic properties associated with the formation and decay of the intermediate observed in the reaction of the binary E-(NADH) complex with the intense substrate chromophore trans-4-(N,N-dimethylamino) cinnamaldehyde (DACA), lambda/sub max/ 398 nm (H/sub 2/O). All the metal ion subsituted enzymes were found to form intermediates with red-shifted spectra upon reaction with DACA and NADH. The magnitudes of (1) the red shifts, (2) the specific rate constants (k/sub off/) for dissociation of DACA from the intermediate, and (3) the hydride transfer rate constants (k /sub H/) were found to correlate with the expected order of the Lewis acid acidities (assuming tetrahedral coordination); i.e., Co(II) > Ni(II) greater than or equal to Zn(II) >> Cd(II). The k/sub H/ value for the Co(II)-E was found to be 1.4-fold greater than the value of 7.2 s/sup -1/ for Zn(II)-E, while the Cd(II)-E was40-fold lower. It is concluded that the close similarities in kinetic properties exhibited by Co(II)-, Ni(II)-, and Zn(II)-enzymes arise from the close similarities in coordination geometries and Lewis acid strengths which lead to highly similar transition states for the processes ofmore » intemediate formation and decay. (JMT)« less