Abstract
Temperature studies have indicated that from 0 to 37 degrees, the time-dependent inactivation of mitochondrial malate dehydrogenase from porcine heart by pyridoxal 5-phosphate (pyridoxal-5-P) is biphasic. The initial phase of the inactivation is reversible but can be made irreversible by reduction with sodium borohydride. The reduced pryidoxal-5-P-enzyme adduct exhibits a new absorbance maximum at 325 nm and a fluorescence emission at 392 nm when excited at 325. The irreversible second phase of the inactivation is accompanied by the appearance of a new 325-nm absorbance maximum, in the absence of reduction, and a fluorescence emission centered about 390 to 400 nm when excited at 325. The evidence presented suggests the formation of a Schiff base between pyridoxal-5-P and a nucleophilic residue, most likely lysine, of malate dehydrogenase during the first phase of inactivation. An X-azolidine-like structure, a further derivative of the Schiff base, possessing spectral properties consistent with the reported data, may be formed during the second phase; this presumably involves a second nucleophilic residue of the enzyme, implicating the action of pyridoxal-5-P as a bifunctional reagent in this instance. The presence of the coenzyme, NADH, protects the enzyme from inactivation, suggesting that pyridoxal-5-P interacts at or near the malate dehydrogenase active center. Simultaneous binding studies using pyridoxal-5-P with known malate dehydrogenase competitive inhibitors AMP, ADP, and nicotinamide indicate that the pyridoxal-5-P modification occurs in the general area of the ADP portion of the coenzyme binging site. Furthermore, the presence of nicotinamide enhances pyridoxal-5-P binding to and inactivation of malate dehydrogenase.
Highlights
L-malate were purchased from Sigma Chemical Company; nicotinamide was obtained from Aldrich Chemical Company
Mitochondrial malate dehydrogenase was purified from acetone powders of fresh pig hearts as previously described by Gregory et al
One can observe an initial drop in malate dehydrogenase activity in the illustrated inactivation immediately upon the addition of pyridoxal-5’-P
Summary
Materials-Pyridoxal-5’.P, sodium borohydride, adenosine 5’-monophosphate, adenosine 5’-diphosphate, NAD+, NADH, andL-malate were purchased from Sigma Chemical Company; nicotinamide was obtained from Aldrich Chemical Company. L-malate were purchased from Sigma Chemical Company; nicotinamide was obtained from Aldrich Chemical Company. Sephadex G-25 was purchased from Pharmacia Fine Chemicals. Mitochondrial malate dehydrogenase was purified from acetone powders of fresh pig hearts as previously described by Gregory et al. Recording A340 on a Unicam SP1800 UV snectrophotometer &&pped with an AR25 Linear Recorder and a’cell compartment thermostatted at 25”. Enzyme Inactivations-Time-dependent inactivations of malate dehydrogenase by pyridoxal-5’-P were carried out in 50 mM sodium phosphate buffer (pH 7.5) at an enzyme concentration of 2 mg per ml (a molecular weight of 70,000 was used for calculations of molar concentrations). Data used in calculations of pseudo-first order rate constants were plotted using a standard linear least squares fit
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