OVEREXPRESSION AND CHARACTERIZATION OF THE HUMAN MITOCHONDRIAL AND CYTOSOLIC BRANCHED-CHAIN AMINOTRANSFERASES

We have developed overexpression systems for the human branched-chain aminotransferase isoenzymes. The enzymes function as dimers and have s ubstrate specificity comparable with the rat enzymes, The human cytoso lic enzyme appears to turn over 2-5 times faster than the mitochondria l enzyme, and there may be anion and cation effects on the kinetics of both enzymes. The two proteins demonstrate similar absorption profile s, and the far UV circular dichroism spectra show that no global struc tural changes occur when the proteins are converted from the pyridoxal to pyridoxamine form, On the other hand, the near UV circular dichroi sm spectra suggest differences in the local environment surrounding ty rosines within these proteins, Both enzymes require a reducing environ ment for maximal activity, but the mitochondrial enzyme can be inhibit ed by nickel ions in the presence of reducing agents, while the cytoso lic enzyme is unaffected, Chemical denaturation profiles of the protei ns show that there are differences in structural stability, Titration of -SH groups with 5,5'-dithiobis(2-nitrobenzoic acid) suggests that n o disulfide bonds are present in the mitochondrial enzyme and that at least two disulfide bonds are present in the cytosolic enzyme, Two -SH groups are titrated in the native form of the mitochondrial enzyme, l eading to complete inhibition of activity, while only one -SH group is titrated in the cytosolic enzyme with no effect on activity, Although these proteins share 58% identity in primary amino acid sequence, the local environment surrounding the active site appears unique for each isoenzyme.