An enzymatically inactive variant of human lactate dehydrogenase-LDHBGUA-1 Study of subunit interaction

Abstract

The LDHBGUA-1 variant is an electrophoretic variant which is enzymatically inactive. It is only detectable because of its ability to form heterotetramers with A and/or active B subunits and alter the electrophoretic pattern, although all evidence suggests the B-GUA-1 subunits are always enzymatically inactive. All tetrameric combinatious of active plus inactive subunits including either an A or active B plus three inactive B subunits possess enzymatic activity. The heterotetramers composed of A and B-GUA-1 subunits are more thermostable than A4 homotetramers but less thermostable than normal AB heterotetramers. The AB-GUA-1 heterotetramers composed of active A and inactive B subunits have a Km for pyruvate, for lactate and for NADH which is similar to that observed for normal AB heterotetramers. The substrate specificity of the A plus normal B heterotetramer and the A plus variant B heterotetramer with [alpha]-hydroxybutyrate or the acetylpyridine analog of NAD as substrate are similar, while differences between the normal and variant erythrocyte isozymes are observed when glyoxalate is the substrate. Interaction with the B-GUA-I subunit reduces the sensitivity of the A subunit to urea inhibition (independent of dissociation), as does the active B subunit, but the inactive B subunit does not modulate the inhibition by oxalate. Although a single active subunit in a tetrameric conformation is sufficient for enzymatic activity, many of the kinetic properties of the lactate dehydrogenase molecule reflect the tetrameric structure rather than the sum of independent subunits. Thus communication among the subunits must exist and conformational changes which affect the catalytic properties of the enzyme (-lactate:NAD+ oxidoreductase, EC 1.1.1.27) must occur during tetramer formation.