FUNCTIONAL EXPRESSION OF C-TERMINALLY TRUNCATED HUMAN MONOAMINE-OXIDASE TYPE-A IN SACCHAROMYCES-CEREVISIAE

The deduced amino acid sequence of human liver monoamine oxidase type A was analyzed with secondary structure programs. These analyses and c omparison to other flavoproteins identified a single potential transme mbrane hydrophobic peptide at the C-terminus suggesting that this pept ide is a membrane anchor and that the remainder of the protein constit utes a soluble domain. Truncation of the C-terminus by 24 amino acids which are inclusive of the putative transmembrane peptide, however, ga ve a protein which exhibited solubility properties substantially simil ar to the wild type enzyme. This result indicates that the hydrophobic behavior of monoamine oxidase type A is due to more complex features than a single transmembrane anchor. The mutant enzyme expressed in yea st appears to form a disulfide bond which reduces catalytic effciency by up to 90%. Full activity, however, can be recovered by incubation w ith dithiothreitol, suggesting that in the wild type enzyme the amino acid residues deleted in the mutant protein protect two cysteine resid ues (those involved in the formation of the disulfide bond in the muta nt) from oxidation and that the deleted residues are in close proximit y to the active site. The activation experiments indicated that the de leted amino acids do not contribute any catalytic residues to the acti ve site.