Date of Award

2000

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Gary Winston

Abstract

The interaction of ADH with C-nitroso, N-nitroso and peroxide substrates was investigated. To better elucidate the mechanism of these interactions, cobalt-substituted HLADH was introduced and compared with native HLADH. Both binary and ternary complexes of p-nitrosophenol with Co(II)ADH caused a blue-shift of the d-d transition region of the Co(II)ADH spectrum, indicating direct interaction of the C-nitroso moiety with the metal active site. In addition to this blue shift with NADH, both pNSP and N,N'-dimethyinitrosamine enhance the absorbance of the 643 nm peak of the ADH/NADH/pyrazole ternary complex, which suggests that these compounds compete with pyrazole for ligand association with the metal center. Enzymatic and nonenzymatic reduction of N-nitrosodiethanolamine, N-nitrosoethylurea, and N-nitrosomethylurea was observed, albeit the reaction rates were relatively slow in comparison to the reduction of C-nitroso compounds; however, the general mechanism involving 2 e-transfer steps in the enzymatic reduction was essentially the same. Spectroscopic studies of Co(II)ADH indicate that N-nitrosodiethanolamine and N-nitrosoethylurea react directly with the coordination sphere of the ADH metal center with strong ternary complex formation. Preincubation of HLADH with H2O 2 caused a first-order rate inactivation (k = 0.11 +/- 0.2 min --1) of the enzyme, whereas tert-butylhydroperoxide (tBOOH) and cumene hydroperoxide (CHP) stimulated HLADH activity by 200 and 170%, respectively. The rate and extent of stimulation by tBOOH were strongly reduced by binary complexes with NAD+ or NADH, whose pyrophosphate groups bind to Arg47 and Arg369. tBOOH irreversibly inactivated yeast ADH (YADH). Similar to H2O2 four --SH groups per molecule of YADH were modified by tBOOH. Spectral analysis of Co(II)ADH indicated strong interaction of peroxides with the active site coordination sphere by the loss of LMCT and d-d transition bands. HLADH stimulation is suggested to be due to destabilization of the catalytic Zn-coordination sphere via indirect interaction of tBOOH with the metal center, while inactivation by H2O2 results from direct interaction with the metal center via irreversible modification of active site ligands Cys46 and Cys174 and modification of an anion-binding site formed by Lys228.

ISBN

9780599906303

Pages

196

Share

COinS