Copper amine oxidases possess the unusual ability to generate autocatalytically their organic cofactor, which is subsequently utilized in turnover. This cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), is formed within the active site of these enzymes by the oxidation of a single tyrosine residue. In vitro, copper(II) and oxygen are both necessary and sufficient for the conversion of tyrosine to TPQ. In this study, the biogenesis of TPQ has been characterized in an amine oxidase from Hansenula polymorpha expressed as the apo-enzyme in Escherichia coli. With the WT enzyme, optical absorbances which are copper or oxygen dependent are observed and characterized. Active-site mutants are used to investigate further the nature of these spectral species. Evidence is presented which suggests that tyrosine is activated for reaction with oxygen by liganding to Cu(II). In the following paper in this issue [Schwartz, B., Dove, J. E., and Klinman, J. P. (2000) Biochemistry 39, 3699-3707], the initial reaction of precursor protein with oxygen is characterized kinetically. Taken together, the available data suggest a mechanism for the oxidation of tyrosine to TPQ where the role of the copper is to activate substrate.

A detailed kinetic analysis of oxygen consumption during TPQ biogenesis has been carried out on a yeast copper amine oxidase. O(2) is consumed in a single, exponential phase, the rate of which responds linearly to dissolved oxygen concentration. This behavior is observed up to conditions of maximally obtainable oxygen concentrations. In contrast, no viscosity effect is observed on rate, implicating a high K(m) for O(2). Binding of oxygen appears to occur faster than its consumption and to result in displacement of the precursor tyrosine onto copper to form a charge-transfer species, described in the the preceding paper of this issue [Dove, J. E., Schwartz, B., Williams, N. K., and Klinman, J. P. (2000) Biochemistry 39, 3690-3698). Reaction between this intermediate and O(2) is proposed to occur in a rate-limiting step, and to proceed more rapidly when the tyrosine is deprotonated. This rate-limiting step in cofactor biogenesis does not display a solvent isotope effect and is, thus, uncoupled from proton transfer. Comparisons are drawn between the proposed biogenesis mechanism and that for the oxidation of reduced cofactor during catalytic turnover in the mature enzyme.

The copper-containing yeast methylamine oxidase E406N mutant has an altered consensus sequence surrounding the topaquinone cofactor (residue 405). The mutation has no effect on the final yield of the active-site topaquinone cofactor during biogenesis but causes the enzyme to be inactivated by substrate methylamine [Cai, D., and Klinman, J. P. (1994) Biochemistry 33, 7674-7653]. In this study we show that the inactivation leads to the formation of a covalent adduct, which has a UV/vis spectrum very similar to that of a product Schiff base, an intermediate of topaquinone-catalyzed amine oxidation reactions. The kinetic isotope effects on the second-order rate constant for the inactivation and catalytic turnover are identical, indicating that the two processes share a common intermediate that follows C_H bond cleavage. Resonance Raman spectroscopy provides direct evidence for the accumulation of a neutral product Schiff base species. Removal of excess methylamine leads to recovery of both activity and the native absorption spectrum for E406N, indicating that the cofactor in the inactivated enzyme is chemically competent for hydrolysis. The rate of the reactivation is slow, however; the shortest half-life of the inhibited E406N at 25 degrees C is 5.9 min at pH 6.15. pH effect experiments show that the inactivation and reactivation steps are controlled by a single ionizable group with a pKa of 6.9-7.1; under basic conditions, when this residue is deprotonated, the inactivation is the fastest and the half-life of the inhibited enzyme is the longest. On the basis of the available crystal structures of copper amine oxidases, we propose that a histidine residue in the dimer interface is responsible for the observed ionization. In the wild-type enzyme this histidine is kept protonated by virtue of Glu at position 406. Unlike methylamine, the larger substrates ethylamine and benzylamine give normal turnover with E406N. Disruption of structure at the subunit interface in E406N may allow a rotation of the relatively small topa-product Schiff base complex (formed from methylamine) away from the active-site base to a conformation that is incompetent toward hydrolysis.

We have generated monoclonal antibodies against nuclear proteins from the yeast Saccharomyces cerevisiae. The monoclonal antibodies react with proteins of 47 and 49 kDa on immunoblots and with partially overlapping sets of proteins on two-dimensional nonequilibrium pH gradient electrophoresis-SDS blots. Immunofluorescence localization shows a nuclear staining pattern. Immunoscreening a yeast expression library yielded five independent full-length clones of two open reading frames from chromosome IV, corresponding to YDL182w (LYS20) and YDL131w in the Saccharomyces genome data base. These two open reading frames are predicted to encode homocitrate synthase isozymes of 47 and 49 kDa, respectively. A clone carrying YDL182w was sequenced in its entirety and directs the expression of a 47-kDa protein in Escherichia coli. A clone carrying YDL131w expresses a 49-kDa protein in E. coli. Yeast grown in minimal medium plus lysine show significant reductions in nuclear immunofluorescence staining. Cell fractionation studies localize the 47-and 49-kDa proteins to the nucleus. Nuclear fractionation studies reveal that a portion of the 47-and 49-kDa proteins can only be extracted with DNase digestion and high salt. The localization of homocitrate synthase to the nucleus is unexpected given previous reports that homocitrate synthase is present in mitochondria and the cytoplasm in S. cerevisiae.In yeast, higher fungi, and euglenids, lysine is synthesized via the ␣-aminoadipate pathway, which is only found in these organisms (1). Homocitrate synthase catalyzes the first committed reaction in this pathway and is thought to be an important site of control of metabolic flow. In Saccharomyces cerevisiae, two isozymes have been identified by isoelectric focusing of purified enzyme preparations (2). Both isozymes are feedback inhibited by lysine, but only one is transcriptionally repressed by lysine (2).Genes for the homocitrate synthase isozymes have not been identified until recently, despite extensive genetic analyses of lysine auxotrophs, which have revealed most of the enzymeencoding (LYS) genes required in this pathway. During the sequencing of chromosome IV of S. cerevisiae, an open reading frame (ORF) 1 was identified that encoded a protein with significant homology to homocitrate synthase from other yeasts (3). This ORF is designated YDL182w in the Saccharomyces genome data base (GenBank™ accession number X83276, ORF D1298). Ramos et al. (4) have disrupted this gene, examined the effects on lysine production and levels of homocitrate synthase enzymatic activity, and named this gene LYS20. The subcellular localization of enzymes of the ␣-aminoadipate pathway has been investigated in S. cerevisiae, and the enzymes for the first half of the pathway have been reported to be located in the mitochondrion (reviewed in Ref. 5). Two reports place homocitrate synthase from S. cerevisiae in mitochondria (6, 7). Jaklitsch and Kubicek (8) reported that homocitrate synthase from Penicillium chrysogenum is present in the mitochondrion and ...