Tyrosine hydroxylase (TyrH) catalyzes the conversion of tyrosine to dihydroxyphenylalanine (DOPA), the rate-limiting step in the biosynthesis of dopamine. Four mutations in the TyrH gene have recently been described in cases of autosomal recessive DOPA-responsive dystonia (Swaans et al., Ann Hum Genet 2000;64:25-31). All four are predicted to result in changes in single amino acid residues in the catalytic domain of the protein: T245P, T283M, R306H, and T463M. To determine the effects of these mutations on the molecular properties of the enzyme, mutant proteins containing the individual single amino acid changes have been expressed in bacteria and purified. Only the T283M mutation results in a decrease in the enzyme k(cat) value, while the T245P enzyme has a slightly higher value than the wild-type enzyme. The only case in which a K(m) value for either tyrosine or tetrahydrobiopterin is perturbed is the T245P enzyme, for which the K(m) value for tyrosine has increased about 50%. In contrast to the minor effects of the mutations on enzyme activity, the stability is decreased significantly by the mutations. The R306H and T283M enzymes are the least stable, losing activity 30- and 50-fold more rapidly than the wild-type enzyme. The apparent T(m) value for unfolding was decreased by 3.9, 8.2, and 7.2 degrees for the T245P, R306H, and T463M enzymes, while the T283M enzyme was too unstable for measurement of a T(m) value. The results establish that the physiological effects of the mutations are primarily due to the decreased stability of the mutant proteins rather than decreases in their intrinsic activities.

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In mammalian cells, the flavoprotein polyamine oxidase catalyzes a key step in the catabolism of polyamines, the oxidation of N1-acetylspermine and N1-acetylspermidine to spermidine and putrescine, respectively. The mechanism of the mouse enzyme has been studied with N1,N12-bisethylspermine (BESPM) as a substrate. At pH 10, the pH optimum, the limiting rate of reduction of the flavin in the absence of oxygen is comparable to the k cat value for turnover, establishing reduction as rate-limiting. Oxidation of the reduced enzyme is a simple second-order reaction. No intermediates are seen in the reductive or oxidative half-reactions. The k cat value decreases below a pK a of 9.0. The k cat /K m value for BESPM exhibits a bell-shaped pH profile, with pK a values of 9.8 and 10.8. These pK a values are assigned to the substrate nitrogens. The rate constant for the reaction of the reduced enzyme with oxygen is not affected by a pH between 7.5 and 10. Active site residue Tyr430 is conserved in the homologous protein monoamine oxidase. Mutation of this residue to phenylalanine results in a 6-fold decrease in the k cat value and the k cat /K m value for oxygen due to a comparable decrease in the rate constant for flavin reduction. This moderate change is not consistent with this residue forming a tyrosyl radical during catalysis.The polyamines spermine, spermidine, and putrescine are ubiquitous in cells. Higher concentrations are found in rapidly growing cells (1-3), and compounds which deplete polyamines from cells inhibit cell growth (2). These observations have led to the general conclusion that polyamines are essential for cell growth, although their specific role in the cell is still a matter of discussion. Consequently, a variety of polyamine analogues have been examined as anticancer drugs (4-7); a number of clinical trials are underway, and analogues with cytotoxic potential have been developed (8,9). The metabolic pathways for synthesis and degradation of polyamines are generally conserved (1). In mammals, the biosynthetic pathway involves decarboxylation of ornithine to putrescine by ornithine decarboxylase, extension of putrescine to spermidine by spermidine synthase using decarboxylated S-adenosylmethionine as the propylamine donor, and a subsequent extension of spermidine with another propylamine moiety to form spermine catalyzed by the enzyme spermine synthase. Depletion of spermine and spermidine from the cell involves the action of two enzymes: spermidine/spermine N1-acetyltransferase converts spermine and spermidine to the respective N1-acetylated compound, and polyamine oxidase oxidizes N1-acetylspermidine and N1-acetylspermine to putrescine and spermidine, respectively, and 3-acetamidopropanal (Scheme 1). While polyamine oxidase can also oxidize spermine, the enzyme strongly prefers N1-acetylated polyamines as substrates (10). Instead, the related enzyme spermine oxidase is responsible for direct oxidation of † This work was supported in part by grants from the NIH (R01 GM58698) and The Welch Fo...

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Protein C inhibitor is a heparin-dependent serine protease inhibitor present in plasma at about 0.08 mumol l-1. Protein C inhibitor inhibits activated protein C and other coagulation factors. Previously, we described the presence of high protein C inhibitor levels in human semen (3.1 mumol l-1) and showed potential roles of the inhibitor in human reproduction. Here, we show that protein C inhibitor is present in an active form in follicular fluid at about 0.1 mumol l-1 and that purified, functionally active human plasma-derived and inactive, semen-derived protein C inhibitor and a synthetic peptide derived from its sequence inhibited both binding and penetration of zona-free hamster oocytes by human sperm. The binding inhibition by protein C inhibitor was dose dependent, with 50% inhibition at 0.037 mumol l-1 inhibitor (45 +/- 17 sperm per egg versus 90 +/- 23 in control experiments). The inhibitor also blocked in a dose-dependent manner the penetration of zona-free hamster eggs by human sperm (20 +/- 7% fertilized eggs at 0.1 mumol l-1 protein C inhibitor versus 55 +/- 10% in control experiments). Polyclonal antiprotein C inhibitor or antipeptide antibodies partially abolished the effect of protein C inhibitor and peptide on the inhibition of the binding and penetration of zona-free hamster oocytes by human sperm. The effect of the protein C inhibitor was not dependent on its antiprotease activity since purified semen-derived protein C inhibitor which did not have antiprotease activity gave comparable results. We conclude that protein C inhibitor may be involved in human reproduction at several steps, including the fertilization process.