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Pig kidney dopa decarboxylase (DDC) expressed in Escherichia coli is a homodimeric enzyme containing one catalytically active pyridoxal 5'-phosphate active site per subunit. In addition to catalyzing the decarboxylation of -aromatic amino acids, DDC also reacts with 5-hydroxytryptamine (5-HT), converting it to 5-hydroxyindolacetaldehyde and ammonia. These products have been identified by means of the enzymes alcohol dehydrogenase and glutamate dehydrogenase, together with high performance liquid chromatographic and mass spectroscopic analysis. The Kcat and Km values of this reaction were determined to be 0.48 min-1 and 0.47 mM, respectively. The NaBH4-reduced enzyme does not catalyze this reaction. Concurrent with this reaction, 5-HT inactivates DDC in both a time- and concentration-dependent manner and exhibits saturation of the rate of inactivation at high concentrations, with Ki and Kinact values of 0.40 mM and 0.023 min-1, respectively. Protection from inactivation by 5-HT was observed in the presence of the active site-directed inhibitor 3,4-dihydroxy-D-phenylalanine. Inactivation with [2-14C]5-HT results in the incorporation of 1 mol of label/enzyme subunit. Taken together, these findings indicate that 5-HT is both a substrate and a mechanism-based inactivator with a partition ratio for product formation versus inactivation of 21. The absorbance, CD, and fluorometric features of 5-HT-inactivated DDC have also been characterized. A speculative mechanism for the reaction and inactivation consistent with the experimental findings is presented.

Saposins A, B, C, and D are a group of homologous glycoproteins derived from a single precursor, prosaposin, and apparently involved in the stimulation of the enzymatic degradation of sphingolipids in lysosomes. All saposins have six cysteine residues at similar positions. In the present study we have investigated the disulfide structure of saposins B and C using advanced mass spectrometric procedures. Electrospray analysis showed that deglycosylated saposins B and C are mainly present as 79- and 80-residue monomeric polypeptides, respectively. Fast atom bombardment mass analysis of peptide mixtures obtained by a combination of chemical and enzymatic cleavages demonstrated that the pairings of the three disulfide bridges present in each saposin are Cys4-Cys77, Cys7-Cys71, Cys36-Cys47 for saposin B and Cys5-Cys78, Cys8-Cys72, Cys36-Cys47 for saposin C. We have recently shown that saposin C interacts with phosphatidylserine-containing vesicles inducing destabilization of the lipid surface (Vaccaro, A. M., Tatti, M., Ciaffoni, F., Salvioli, R., Serafino, A., and Barca, A. (1994) FEBS Lett. 349, 181-186); this perturbation promotes the binding of the lysosomal enzyme glucosylceramidase to the vesicles and the reconstitution of its activity. It was presently found that the effects of saposin C on phosphatidylserine liposomes and on glucosylceramidase activity are markedly reduced when the three disulfide bonds are irreversibly disrupted. These results stress the importance of the disulfide structure for the functional properties of the saposin.

10-Formyltetrahydrofolate dehydrogenase catalyzes the NADP(+)-dependent oxidation of 10-formyltetrahydrofolate to CO2 and tetrahydrofolate. Previous studies have shown that the enzyme binds the physiological pentaglutamate form of tetrahydrofolate product so tightly that it remains bound during size exclusion chromatography (Cook, R. J., and Wagner, C. (1982) Biochemistry 21, 4427-4434). In addition to the dehydrogenase activity, the enzyme from rat liver has been reported to exhibit both 10-formyltetrahydrofolate hydrolase and aldehyde dehydrogenase activities (Cook, R. J., Lloyd, R. S., and Wagner, C. (1991) J. Biol. Chem. 266, 4965-4973). We have purified the enzyme from rabbit liver and found that it catalyzes the same three reactions with similar kinetic constants and that it is a 99-kDa homotetramer, as reported previously for the rat and pig enzymes. Previous studies have suggested that the enzyme is composed of three domains and has separate folate binding sites for the dehydrogenase and hydrolase activities. We have investigated the domain structure of the rabbit enzyme. Differential scanning calorimetry reveals two thermal transitions, indicating the presence of two independently folded domains. The pentaglutamate form of tetrahydrofolate and NADP+ each stabilize one of the thermal transitions, showing that these ligands bind to separate domains. Limited proteolytic digestions by several proteases cleave the enzyme in a linker region between the two domains. After proteolytic cleavage, the domains no longer remain associated and do not catalyze the 10-formyltetrahydrofolate dehydrogenase reaction. Isolation and characterization of the intact domains revealed that the N-terminal domain only catalyzes the NADP(+)-independent 10-formyltetrahydrofolate hydrolase activity and the C-terminal domain only catalyzes the NADP(+)-dependent aldehyde dehydrogenase activity. The kinetic constants of these isolated domains are similar to those of the intact enzyme. Binding studies on the native enzyme using fluorescence and isothermal titration calorimetry indicated that the enzyme binds one molecule of tetrahydrofolate and two molecules of NADP+ per tetramer. Dissociation constants for both ligands were also determined by these methods.

The primary sequence of 5,10-methenyltetrahydrofolate synthetase from rabbit liver was determined by amino acid sequencing of the purified enzyme. The enzyme contains 201 amino acid residues with a predicted mass of 22,779 Da. The enzyme is located in the cytosolic fraction of liver homogenates. Carbodiimide-activated 5-formyltetrahydropteroylmonoglutamate and the pentaglutamate form of the substrate both irreversibly inactivate the enzyme by forming a covalent bond to Lys-18. Non-activated 5-formyltetrahydropteroylpentaglutamate protected against this inactivation. Substrate specificity studies showed that increasing the number of glutamate residues from zero to five on 5-formyltetrahydropteroate results in a 2 order of magnitude increase in the affinity of the substrate for the enzyme but only a 3-fold increase in the value of Vmax.

The complete amino acid sequence of mitochondrial serine hydroxymethyltransferase from rabbit liver was determined. The sequence was obtained from analysis of peptides isolated from chymotryptic, cyanogen bromide, and limited acid cleavages of the protein. The enzyme consists of four identical subunits, each of 475 residues, i.e. 8 residues shorter than the subunit of the corresponding cytosolic isoenzyme. The sequences of the two rabbit proteins are easily aligned, provided a gap of 5 residues near the amino terminus and a gap of 3 residues near the carboxyl terminus are included in the mitochondrial sequence. The overall degree of identity between the two isoenzymes is 61.9%, whereas the structural identity of each eukaryotic isoenzyme with the corresponding Escherichia coli enzyme is about 40%. The rabbit isoenzymes are about 70 residues longer than the E. coli enzyme, with one-half of these residues accounted for by insertions in both isoenzymes near their carboxyl terminus. Predictions of secondary structure and calculations of hydropathy profiles are also presented, suggesting an even more extensive degree of identity in the three-dimensional folding of the three proteins, in accord with the known similarity of their catalytic properties. Evidence was obtained for the existence of additional molecular forms of the mitochondrial protein, differing in the absence of some amino acid residues at the amino terminus of the polypeptide chain.