How the loss of several residues, at the level of one interglobule junction, modulates the lactate dehydrogenase activity of yeast flavocytochrome b2: a study of the nicked enzymes resulting from clostripain and trypsin action.

The native chain of flavocytochrome b2 is folded into three globules linked together by two protease-sensitive bridges "a" and "cd". We show in this paper that zone "a" of H-flavocytochrome b2 is the first to be cleaved under clostripain action. The alpha c and beta c fragments thus formed are homologous to alpha T and beta'T trypsic fragments. The remaining activities of the resulting (alpha c beta c) and alpha T beta'T) forms are only 25 per cent and 4 per cent of the native flavocytochrome b2 one. The study of the catalytic properties of (alpha c beta'T) and (alpha T beta c) species resulting from the crossed reassociation of the isolated fragments show that the beta type fragment plays a critical role in the catalytic process. A dramatic activity decrease may be correlated with the loss of 6 amino acid residues at the N-terminal of beta c. Our best hypothesis is that these amino acids are involved in the active site, which may be located in the contact zone between alpha and beta. These results are in agreement with previous results obtained in this laboratory which showed the necessity of both alpha T and beta'T fragments for the correct conformation of the flavin binding site.