Methods for Characterizing TPQ-Containing Proteins

Abstract

Publisher Summary The standard 20 amino acid side chains define a repertoire of chemical function rich in potent nucleophiles but notably electrophile poor. Nature overcomes this deficiency by either supplying an exogenous cofactor to an enzyme, such as pyridoxal phosphate (PLP), pyrroloquinoline quinine (PQQ), or a metal ion, or by modifying the existing amino acid side chains in situ to supply an active site with reactive carbonyls. The latter strategy has certain advantages, because it obviates the need for transport and insertion of a reactive species across cellular space and into an enzyme. In 1990, the first of what has been shown to be a series of covalently linked, side chain–derived cofactors was discovered and definitively described as 2,4,5 trihydroxyphenylalanine quinone (topaquinone, or TPQ). TPQ is generated post-translationally in a spontaneous process that depends only on the active site Cu and O 2 . Two observations first indicated that this was true. First, Hansenula polymorpha (HPAO) heterologously expressed in S. cerevisiae , a host with no endogenous copper amine oxidases (CAOs), has fully processed and functional TPQ, suggesting that special host-produced factors are not necessary for TPQ formation. Second, CAO expressed in hosts without adequate Cu(II) supplementation in the growth medium is purified with less than an equivalent of Cu(II) per subunit. Work with CAOs has progressed to the stage where these enzymes can be used as virtual laboratories for exploring detailed mechanistic questions. Such a detailed understanding will complement efforts to understand broader questions about CAOs, their roles in nature, and their place in the wider family of quinoproteins.