Involvement of Serine 74 in the Enzyme-Coenzyme Interaction of Rat Liver Mitochondrial Aldehyde Dehydrogenase

Rout, Ujjwal K.; Weiner, Henry

doi:10.1021/bi00196a013

Cited by 17 publications

(35 citation statements)

References 26 publications

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“…Replacement of conserved residues by neutral amino acids had less effect on the activity than when oppositely charged amino acids were used (Table I). No major changes in the K m for propionaldehyde was found with the mutant enzymes, when compared with the native enzyme, as was observed for other mutants we previously characterized (11,12,21). The exception was with C302S, where the K m for propionaldehyde increased several thousandfold (10).…”

Section: Expression and Purification Of Human Native And Mutantsupporting

confidence: 78%

“…Pre-steady State Burst of NADH Formation-The pre-steady state burst magnitude of NADH formation was determined with a Hitachi 2000 Fluorescence Spectrophotometer (12,21). Enzyme and NAD ϩ were incubated in 100 mM sodium phosphate (pH 7.4) to establish a fluorescence base line.…”

Section: Materials-nadmentioning

confidence: 99%

“…The extrapolated line intersecting at time zero gave the magnitude of the burst of NADH formation. By calibrating the fluorometer with various concentrations of NADH, it was possible to calculate the moles of NADH produced prior to the steady state rate of NADH formation (12,14). Determination of Protein Concentration-The protein concentration was determined with the Bio-Rad protein assay kit, using bovine serum albumin as a standard.…”

Section: Materials-nadmentioning

confidence: 99%

“…The dissociation constant (K ia ) for NAD ϩ with ALDH2 was determined by bisubstrate kinetic analysis (11,12,21). Several concentrations of NAD ϩ and propionaldehyde were used for the analysis of the data.…”

Section: Expression and Purification Of Human Native And Mutantmentioning

confidence: 99%

“…It was shown later by site-directed mutagenesis studies that Cys 302 is a nucleophile (10) and Glu 268 (11) functions as a general base during catalysis. However, Ser 74 (12) and Cys 49 /Cys 162 (10) were found to be not essential for the catalytic reaction.…”

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confidence: 95%

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The Potential Roles of the Conserved Amino Acids in Human Liver Mitochondrial Aldehyde Dehydrogenase

Sheikh

Hurley

et al. 1997

Journal of Biological Chemistry

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The sequence alignment of all known aldehyde dehydrogenases showed that only 23 residues were completely conserved (Hempel, J., Nicholas, H., and Lindahl, R. (1993 Oxidation of toxic aldehydes to their corresponding acids is primarily catalyzed by aldehyde dehydrogenase (ALDH).1 During the last two decades, several ALDHs from different organisms were discovered. By aligning the sequences of 16 known ALDHs, it was found that only 23 amino acids were completely conserved (1). More recently, Vasiliou et al. (2) classified 26 mammalian ALDHs based on divergent evolution and reported that same residues were conserved. We undertook a mutagenesis approach to investigate the role of the conserved residues in the human mitochondrial ALDH, which possessed a functional side chain. Since completion of the study, the threedimensional structure of the corresponding beef liver enzyme has been determined to 2.65 Å (3).Prior to the advent of molecular biological techniques, chemical modifications were used to identify the components of the active site of the enzyme. Classical sulfhydryl reagents inactivated the enzyme (4 -6). Iodoacetamide was shown to modify Cys 302 in human ALDH (4) while using protection studies; our laboratory reported that Cys 49 was a component of the active site of the horse liver enzyme (7). Two other residues were identified by chemical modifications as being possibly involved in the catalytic process. These were Glu 268 and Ser 74 in the human (8) and sheep liver (9) enzymes, respectively. It was shown later by site-directed mutagenesis studies that Cys 302 is a nucleophile (10) and Glu 268 (11) functions as a general base during catalysis. However, Ser 74 (12) and Cys 49 /Cys 162 (10) were found to be not essential for the catalytic reaction.The kinetics of ALDH was found to follow an ordered sequential mechanism where NAD ϩ binds first followed by aldehyde (13). The reaction involves both acylation and deacylation steps during the oxidation of aldehyde to acid, or hydrolysis of nitrophenyl acetate. It was proposed that deacylation (k 7 ) (Fig. 1) was rate-limiting for horse liver ALDH2 (13, 14) for the dehydrogenase reaction, while acylation (k 3 ) was rate-limiting for the esterase reaction.Here we report the properties of human ALDH2 mutant enzymes produced by replacing the conserved amino acids possessing a reactive side chain with different residues. The purpose of this study is to understand the potential role of the conserved residues in the catalytic mechanism of ALDH and to relate the effects to the now known structure. In the accompanying paper, a detailed analysis of the Lys 192 and Glu 399 mutants that caused a change in the rate-limiting step of the enzyme will be presented (15). EXPERIMENTAL PROCEDURES Materials-NADϩ and NADH were purchased from Sigma; Sequenase version 2.0 kit was obtained from United States Biochemical Corp.; propionaldehyde, chloroacetaldehyde, and p-nitrophenyl acetate were from Aldrich; Magic Minipreps DNA purification system and T4 DNA ligase were from Promega Corp...

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Section: Expression and Purification Of Human Native And Mutantsupporting

confidence: 78%

Section: Materials-nadmentioning

confidence: 99%

Section: Materials-nadmentioning

confidence: 99%

Section: Expression and Purification Of Human Native And Mutantmentioning

confidence: 99%

mentioning

confidence: 95%

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