Substrate-induced conformational changes in lactate dehydrogenase. Proteolysis of the immobilized enzyme in the presence of specific substrates.

We report here a new approach to the study of the conformation of enzymes in the presence of specific substrates. Rabbit muscle lactate dehydrogenase was attached to CL-Sepharose via a cleavable spacer arm (-NH-(CH2)6NHCO(CH2)2SS(CH2)2CO-). The bound lactate dehydrogenase was digested with subtilisin BPN' in the presence of substrates of lactate dehydrogenase. The use of a flow system permits the maintenance of saturating levels of substrates. Proteolysis was followed by loss of activity of the enzyme column. The time course of proteolysis in the presence of either NADH, NAD+, or pyruvate alone did not differ from the control. However, when NADH and pyruvate were present simultaneously, the enzyme became more susceptible to proteolysis. The initial rate of proteolysis was increased by 40%. The abortive ternary complex (lactate dehydrogenase - NAD+ - pyruvate) also showed an increase in susceptibility to proteolysis. These findings clearly show that the productive ternary complex (lactate dehydrogenase - NADH - pyruvate) is conformationally different from the apoenzyme and binary complexes under optimal catalytic conditions.