Involvement of serine 74 in the enzyme-coenzyme interaction of rat liver mitochondrial aldehyde dehydrogenase.
It has been suggested that the active site nucleophile in sheep liver aldehyde dehydrogenase was not a cysteine residue but was a serine located at position 74 [Loomes, K. M., Midwinter, G. G., Blackwell, L. F., & Buckley, P. D. (1990) Biochemistry 29, 2070-2080]. This enzyme form has not yet been cloned and expressed, but since the rat liver mitochondrial enzyme has been and shares 70% sequence homology with other cytosolic aldehyde dehydrogenases, the residue in the rat enzyme was converted into an alanine to test for the necessity of a hydroxyl group at that position. The recombinantly expressed mutant enzyme possessed 10% catalytic activity, but the Km for NAD increased from 10 to 1900 microM while the Kms for various aldehydes were unchanged. Kinetic analysis revealed that the dissociation constant for NAD also increased in the mutant as did k1, the on velocity for NAD binding. The mutant enzyme bound poorly to an AMP-Sepharose column and did not interact as well with NADH, as determined by fluorescence enhancement binding studies, or with ADP-ribose, a competitive inhibitor. Pulse-chase analysis showed that the mutant was as stable as was the recombinantly expressed native enzyme. It was less stable to heat denaturation at 50 degrees C (half-life of 1 min compared to 4). Converting the alanine to a cysteine or a threonine did not restore native-like properties of the enzyme. These mutants had kinetic properties very similar to those of the alanine mutant.(ABSTRACT TRUNCATED AT 250 WORDS)
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Biochemistry Department, Purdue University, West Lafayette, Indiana 47907-1153.
Weiner
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D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000444:Aldehyde Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D003067:Coenzymes / Q000378:metabolism*; D004795:Enzyme Stability; D004950:Esterases / Q000378:metabolism; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D009154:Mutation; D009243:NAD / Q000096:biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification; D012685:Sepharose / Q000031:analogs & derivatives / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D013379:Substrate Specificity
D003067:Coenzymes; D011994:Recombinant Proteins; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D012694:Serine; C029427:adenosine monophosphate-sepharose; D012685:Sepharose; D000444:Aldehyde Dehydrogenase; D004950:Esterases