Comparative kinetics between matrix-bound lipoamide dehydrogenase and the free enzyme.

Abstract

Lipoamide dehydrogenase (EC 1.6.4.3) has been succesfully linked to a CNBr-activated polysaccharide matrix, Sepharose-4B, under different reaction conditions. The enzyme is probably bound more homogeneously at lower pH values (pH 7.5) than at pH 8.5. Such immobilized preparations yield V values 8-30% of the value of the V of the free enzyme (18,600 mole/min/mole of flavin). A low level of CNBr-activation in combination with substrate protection and a pH of 7.5 during the coupling reaction leads to the most active preparations. The Km values for both substrates increase considerably. The overall kinetic pattern of the matrix-bound enzyme preparations is not different from that of the free enzyme. Both activation by high concentration of lip (SH)2NH2 at low NAD+ levels and a stimulation of the V by increased phosphate concentration in the assay buffer is observed with free and some immobilized enzymes.