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Affinity labeling of a lysine residue in the coenzyme binding site of pig heart mitochondrial malate dehydrogenase



Affinity labeling of a lysine residue in the coenzyme binding site of pig heart mitochondrial malate dehydrogenase



Biochemistry 18(21): 4683-4690



Porcine heart mitochondrial malate dehydrogenase is inactivated with a pseudo-1st-order rate constant of 0.00941 min by 2.1 mM 5'-[p-(fluorosulfonyl)benzoyl]adenosine in 0.02 M sodium barbital buffer, pH 8.0, containing 15% dimethylformamide (DMF). The pseudo-1st-order rate constant for inactivation is linearly dependent on the reagent concentration. The adenosine moiety is crucial, as indicated by the observation that p-(fluorosulfonyl)benzoic acid (which lacks the adenosine group) inactivates the enzyme 37-fold more slowly. Marked protection against this inactivation is provided by NADH and NAD, but not by malate. The Kd for NADH and NAD, calculated from the decrease in the inactivation rate, are 4.6 and 385 .mu.M, respectively, values close to those obtained by previous direct binding measurements. This dimeric enzyme is known to be dissociated to its monomeric form at low protein concentration and low pH. Sedimentation equilibrium and light-scattering studies reveal that the native enzyme retains its dimeric structure in the absence of 15% DMF under the conditions of reaction with 5'-[p-(fluorosulfonyl)benzoyl]adenosine, and gel filtration experiments demonstrate that the state of aggregation of the enzyme is not altered upon modification by this reagent. A plot of the incorporation of [3H]-5'-(p-sulfonylbenzoyl)adenosine vs. loss of enzyme activity is linear throughout the range tested, and extrapolation leads to 0.97 mol of radioactive reagent incorporated/mol of enzyme subunit at 100% inactivation. Fractionation of the acid hydrolysate of the modified enzyme by amino acid analysis or by 2-dimensional TLC and electrophoresis leads to the identification of lysine as the modified amino acid. The presence of a lysine residue at or near the coenzyme binding site of porcine heart mitochondrial malate dehydrogenase is indicated.

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Accession: 014965430

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PMID: 497160

DOI: 10.1021/bi00588a032



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