The long- term objective of this research project is to analyze the kineticproperties of alcohol-metabolizing enzymes. The hypothesis is that humanalcohol dehydrogenases vary in kinetic mechanism and rate-limiting step, and that chronic alcohol consumption perturbs the kinetic mechanism of themicrosomal ethanol-oxidizing system MEOS. Ingested alcohols aremetabolized predominantly by either alcohol dehydrogenases or MEOS. Atleast five alcohol dehydrogenase subunits are expressed in the liver andinclude alpha, beta, and gamma Class I, eta Class II, and chi ClassIII. A sixth class of alcohol dehydrogenase, sigma, is expressed in thestomach and may be involved in first-pass metabolism. These isoenzymes, with natural substitutions at positions 48 and 93 in the substrate bindingsite, differ considerably in substrate specificity. Natural substitutionsof His-47 or Cys-369 for Arg-47 and Arg-369 in the beta subunit producesisoenzymes that differ dramatically in coenzyme binding affinity. The MEOSis a membrane-bound multi-enzyme complex containing P450 isoenzymes. Long- term alcohol exposure in rats leads to an increase in a hepatic ethanol- induced isoenzyme, P450 IIE, and alters membrane composition in the cells. These changes may affect the kinetic mechanism ofthe P450 IIE isoenzyme. I have examined by stopped-flow techniques the coenzyme bindingcharacteristics of human beta1beta1 enzymes expressed in E coli withmutations at position 47 I will use this Scientist Development Award todevelop stopped-flow methodsfor examining the coenzyme and substrateapparent binding rate constants and limiting hydride transfer rates ofbeta1beta1 enzymes containing site-specific mutations at position 369 andof stomach sigma-alcohol dehydrogenase. I will develop research techniquesfor using stopped-flow kinetics to study the kinetic mechanism andsubstrate specificity of P450 IIE, and to determine the effects of long- term alcohol exposure on P450 IIE mechanism in microsomal liposomes. Iwill also use site-directed mutagenesis andmolecular graphics to expressand purify enzymes with site-specific mutations at positions 48 and 93, andthen with these mutants construct a lattice structure of substratespecificity with molecular graphics.
Alcohol Dehydrogenase, Alcoholism Alcohol Abuse, Binding Protein, Catalyst, Chemical Substitution, Computer Graphics Printing, Cytochrome P, Endoplasmic Reticulum, Enzyme Complex, Enzyme Mechanism, Enzyme Structure, Enzyme Substrate, Ethanol, Human Tissue, Immunodiffusion, Isozyme, Laboratory Rabbit, Laboratory Rat, Liposome, Liver Metabolism, Membrane Structure, Microsome, Mutant, Nadph Oxidoreductase, Sex Difference, Site Directed Mutagenesis, Stomach, Stop Flow Technique
