STUDIES ON THE CONFORMATIONAL CHANGES OF MITOCHONDRIAL MALATE DEHYDROGENASE IN UREA–PHOSPHATE SOLUTIONS

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Abstract
Pig-heart mitochondrial malate dehydrogenase is gradually inactivated in 4 M urea. During the inactivation, sulfhydryl groups on the protein are exposed in a first-order reaction. The reaction is followed spectrophotometricaily using the sulfhydryl reagent, 5,5′-dithiobis(2-nitrobenzoate) (DTNB). Titration with DTNB in the presence of urea exposes 10 to 12 sulfhydryl groups per molecule of mitochondrial malate dehydrogenase. The enzyme is also inactivated when diluted in water but no sulfhydryl groups are unmasked. The loss of activity and the appearance of sulfhydryl groups in urea solutions do not take place at the same rate.The conformational changes of malate dehydrogenase that occur in urea solutions are partially prevented by inorganic phosphate ions, and less so by the substrates NADH, NAD+, oxalacetate (OAA), and L-malate. The protection against loss of enzyme activity by inorganic phosphate ions is pH-dependent. Both inorganic phosphate and NADH considerably reduce the first-order rate constant for sulfhydryl appearance in 4 M urea. Protection of the enzyme against sulfhydryl appearance in urea solutions by pre-incubation with the substrates indicates that about two sulfhydryl groups per molecule of mitochondrial malate dehydrogenase are involved in substrate binding. Thus, the substrates must keep the active site of the enzyme intact. They either bind to the sulfhydryl groups or prevent the protein molecule from completely unfolding.