Reinvestigation of the phenacyl bromide modification of alpha-chymotrypsin.

The modification of alpha-chymotrysin with phenacyl bromide has been reinvestigated over a wide pH range. Evidence is presented that indicates that the nature of the phenacyl-modified enzymes prepared by this reaction is dependent upon the pH of the reaction medium. The phenacyl alpha-chymotrypsin produced at low pH is most probably the Met-192 phenacylsulfonium salt, as proposed earlier, since it readily undergoes dealkylation using 2-mercaptoethanol. However, the phenacyl-enzyme prepared at neutral pH possesses a much reduced enzymatic activity and does not react with 2-mercaptoethanol to regenerate native alpha-chymotrypsin. In addition, incubation of the Met-192 phenacyl sulfonium enzyme at neutral pH causes a smooth irreversible change to the new phenacyl-enzyme as monitored by changes in enzymatic activity, susceptibility to dealkylation using 2-mercaptoethanol, and ultraviolet difference absorption spectral properties. The stoichiometries of both the low and neutral pH modification reactions have been determined, using [carbonyl-14C]phyenacyl bromide, to be 1 phenacyl group/enzyme molecule. In efforts to obtain information about the nature and mechanism of formation of the phenacyl alpha-chymotrypsin produced at neutral pH, alkylation reactions of modified alpha-chymotrypsins produced by His-57 functionalization with tosylphenylalanine chloromethyl ketone and by Met-192 oxidation to the sulfoxide have been investigated. The combined results of these studies have been initially interpreted in terms of a neutral pH, phenacyl bromide modification resulting in formation of a new modified enzyme via the Met-192 sulfonium salt.     

alpha-Bromo-4-amino-3-nitroacetophenone, a new reagent for protein modification. Modification of the methionine-290 residue of porcine pepsin.

A new coloured reagent for protein modification, alpha-bromo-4-amino-3-nitroacetophenone (NH2BrNphAc), was synthesized. The reagent was found to alkylate specifically the methionine-290 residue of porcine pepsin below pH 3 at 37 degrees C, which lead to a 45% decrease of enzyme's activity towards haemoglobin. The effect of this reagent as well as that of other phenacyl bromides on the activity of pepsin appeared to be a result of steric hindrance caused by the attachment of bulky reagent residue to the edge of the cleft harbouring the enzyme active site. Only marginal reaction with the co-carboxy group of aspartic acid-315 was found under the above conditions. More pronounced esterification of carboxy groups (up to one residue per enzyme molecule) occurred when the pH was shifted to 5.2. The latter modification had no noticeable effect on enzyme activity, thus disproving a previously held assumption that pepsin inactivation by phenacyl bromide is due to the carboxy-group esterification. alpha-Bromo-4-amino-3-nitroacetophenone forms derivatives with characteristic u.v. spectra when it reacts with methionine, histidine, aspartic and glutamic acid residues, and may be recommended as a reagent for protein modification.     

Mutational analysis of the RNA triphosphatase component of vaccinia virus mRNA capping enzyme.

Vaccinia virus mRNA capping enzyme is a multifunctional protein with RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7-) methyltransferase activities. The enzyme is a heterodimer of 95- and 33-kDa subunits encoded by the vaccinia virus D1 and D12 genes, respectively. The N-terminal 60-kDa of the D1 subunit (from residues 1 to 545) is an autonomous domain which catalyzes the triphosphatase and guanylyltransferase reactions. Mutations in the D1 subunit that specifically inactivate the guanylyltransferase without affecting the triphosphatase component have been described (P. Cong and S. Shuman, Mol. Cell. Biol. 15:6222-6231, 1995). In the present study, we identified two alanine-cluster mutations of D1(1-545), R77A-K79A and E192A-E194A, that selectively inactivated the triphosphatase, but not the guanylyltransferase. Concordant mutational inactivation of RNA triphosphatase and nucleoside triphosphatase functions (to approximately 1% of wild-type specific activity) suggests that both gamma-phosphate cleavage reactions occur at a single active site. The R77A-K79A and E192A-E194A mutant enzymes were less active than wild-type D1(1-545) in the capping of triphosphate-terminated poly(A) but could be complemented in vitro by D1(1-545)-K260A, which is inert in nucleotidyl transfer but active in gamma-phosphate cleavage. Whereas wild-type D1(1-545) formed only the standard GpppA cap, the R77A-K79A and E192A-E194A enzymes synthesized an additional dinucleotide, GppppA. This finding illuminates a novel property of the vaccinia virus capping enzyme, the use of triphosphate RNA ends as an acceptor for nucleotidyl transfer when gamma-phosphate cleavage is rate limiting.     

Exceptional stability of a [2Fe-2S] ferredoxin from hyperthermophilic bacterium Aquifex aeolicus

Aquifex aeolicus is the only hyperthermophile that is known to contain a plant- and mammalian-type [2Fe-2S] ferredoxin (Aae Fd1). This unique protein contains two cysteines, in addition to the four that act as ligands of the [2Fe-2S] cluster, which form a disulfide bridge. We have investigated the stability of Aae Fd1 with (wild-type) and without (C87A variant) the disulfide bond, with respect to pH, thermal and chemical perturbation, and compared the results to those for the mesophilic [2Fe-2S] ferredoxin from spinach. Unfolding reactions of all three proteins are irreversible due to cluster decomposition in the unfolded state. Wild-type and C87A Aae Fd1 proteins are extremely stable: unfolding at 20 degrees C requires high concentrations of the chemical denaturant and long incubation times. Moreover, their thermal-unfolding midpoints are 40-50 degrees higher than that for spinach ferredoxin (pH 7). The stability of the Aae Fd1 protein is significantly lower at pH 2.5 than pH 7 and 10, suggesting that ionic interactions play a role in structural integrity. Interestingly, the iron-sulfur cluster in C87A Aae Fd1 rearranges into a transient species with absorption bands at 520 and 610 nm, presumably a linear three-iron cluster, in the high-pH unfolded state.     

Computational identification of significantly regulated metabolic reactions by integration of data on enzyme activity and gene expression

The concentrations and catalytic activities of enzymes control metabolic rates. Previous studies have focused on enzyme concentrations because there are no genome-wide techniques used for the measurement of enzyme activity. We propose a method for evaluating the significance of enzyme activity by integrating metabolic network topologies and genome-wide microarray gene expression profiles. We quantified the enzymatic activity of reactions and report the 388 significant reactions in five perturbation datasets. For the 388 enzymatic reactions, we identified 70 that were significantly regulated (P-value < 0.001). Thirty-one of these reactions were part of anaerobic metabolism, 23 were part of low-pH aerobic metabolism, 8 were part of high-pH anaerobic metabolism, 3 were part of low-pH aerobic reactions, and 5 were part of high-pH anaerobic metabolism.     

S-Adenosylmethionine decarboxylase from human prostate. Activation by putrescine

1. The presence of S-adenosylmethionine decarboxylase in human prostate gland is reported. A satisfactory radiochemical enzymic assay was developed and the enzyme was partially characterized. 2. Putrescine stimulates the reaction rate by up to 6-fold at pH7.5: the apparent activation constant was estimated to be 0.13mm. The stimulation is pH-dependent and a maximal effect is observed at acid pH values. 3. Putrescine activation is rather specific: other polyamines, such as spermidine and spermine, did not show any appreciable effect. 4. The apparent K(m) for the substrate is 4x10(-5)m. The calculated S-adenosylmethionine content of human prostate (0.18mumol/g wet wt. of tissue) demonstrates that the cellular amounts of sulphonium compound are saturating with respect to the enzyme. 5. The enzyme is moderately stable at 0 degrees C and is rapidly inactivated at 40 degrees C. The optimum pH is about 7.5, with one-half of the maximal activity occurring at pH6.6. 6. Several carboxy-(14)C-labelled analogues and derivatives of S-adenosylmethionine were tested as substrates. The enzyme appears to be highly specific: the replacement of the 6'-amino group of the sulphonium compound alone results in a complete loss of activity. 7. Inhibition of the enzyme activity by several carbonyl reagents suggests an involvement of either pyridoxal phosphate or pyruvate in the catalytic process. 8. The inhibitory effect of thiol reagents indicates the presence of ;essential' thiol groups.     

Comparative study of the extracellular laccases from Cerrena unicolor 059 0784 and Pleurotus oastreatus 0432

The laccases of the basidiomycetes Cerrena unicolor 059, C. unicolor 0784, and Pleurotus oastreatus 0432 were assayed comparatively. The laccases were isolated as homogenous preparations with molecular weight 55, 56, and 57 kD, respectively. The three enzymes were found to be glycoproteins. The carbohydrate moiety of the glycoproteins included mannose, galactose, and N-acetylglucosamine. The carbohydrate moiety of the laccases from C. unicolor 059, C. unicolor 0784, and P. oastreatus 0432 accounted for 17, 23, and 24%, respectively. The pH optimum of the enzymes was at 4.0, 3.75, and 5.6, respectively. Thermal stability testing of laccases at 40 degrees C revealed that the C. unicolor 0784 enzyme was characterized by the highest thermal stability (after 172-h incubation the enzyme activity was maintained at a level of 25%). The Michaelis constant (Km) values of the reactions of oxidation of pyrocatechol, hydroquinone, and potassium ferrocyanide catalyzed by the basidiomycete laccases were determined.     

Substrate specificity of 5''-methylthioadenosine phosphorylase from human prostate.

5'-Methylthioadenosine phosphorylase was purified approx. 340-fold from human prostate by using affinity chromatography by Hg-coupled Sepharose. The enzyme, responsible for the breakdown of 5'-methylthioadenosine into adenine and methylthioribose 1-phosphate, was partially characterized. The apparent Km for 5'-methylthioadenosine is 25 microM. It is activated by thiols and shows an absolute requirement for phosphate ions. New analogues of 5'-methylthioadenosine were prepared and their activity as substrates or inhibitors of the reaction was investigated. The replacement of the 6-amino group of the adenine moiety by a hydroxy group, as well as the replacement of N-7 by a methinic radical, resulted in an almost complete loss of activity. Otherwise the replacement of sulphur by selenium, as well as that of the methyl group by an ethyl one, is compatible with the activity as substrate. The positively charged sulphonium group also prevents catalytic interaction with the enzyme. The inhibitory effect of 5'-methylthiotubercidin (competitive) and 5'-dimethylthioadenosine sulphonium salt (non-competitive) was also demonstrated. The reported results suggest three binding sites between the substrate and the enzyme.     

Biochemical characterization of a structure-specific resolving enzyme from Sulfolobus islandicus rod-shaped virus 2

Sulfolobus islandicus rod shaped virus 2 (SIRV2) infects the archaeon Sulfolobus islandicus at extreme temperature (70°C-80°C) and acidity (pH 3). SIRV2 encodes a Holliday junction resolving enzyme (SIRV2 Hjr) that has been proposed as a key enzyme in SIRV2 genome replication. The molecular mechanism for SIRV2 Hjr four-way junction cleavage bias, minimal requirements for four-way junction cleavage, and substrate specificity were determined. SIRV2 Hjr cleaves four-way DNA junctions with a preference for cleavage of exchange strand pairs, in contrast to host-derived resolving enzymes, suggesting fundamental differences in substrate recognition and cleavage among closely related Sulfolobus resolving enzymes. Unlike other viral resolving enzymes, such as T4 endonuclease VII or T7 endonuclease I, that cleave branched DNA replication intermediates, SIRV2 Hjr cleavage is specific to four-way DNA junctions and inactive on other branched DNA molecules. In addition, a specific interaction was detected between SIRV2 Hjr and the SIRV2 virion body coat protein (SIRV2gp26). Based on this observation, a model is proposed linking SIRV2 Hjr genome resolution to viral particle assembly.     

The biochemical characterization of two carotenoid cleavage enzymes from Arabidopsis indicates that a carotenoid-derived compound inhibits lateral branching

Enzymes that are able to oxidatively cleave carotenoids at specific positions have been identified in animals and plants. The first such enzyme to be identified was a nine-cis-epoxy carotenoid dioxygenase from maize, which catalyzes the rate-limiting step of abscisic acid biosynthesis. Similar enzymes are necessary for the synthesis of vitamin A in animals and other carotenoid-derived molecules in plants. In the model plant, Arabidopsis, there are nine hypothetical proteins that share some degree of sequence similarity to the nine-cis-epoxy carotenoid dioxygenases. Five of these proteins appear to be involved in abscisic acid biosynthesis. The remaining four proteins are expected to catalyze other carotenoid cleavage reactions and have been named carotenoid cleavage dioxygenases (CCDs). The hypothetical proteins, AtCCD7 and AtCCD8, are the most disparate members of this protein family in Arabidopsis. The max3 and max4 mutants in Arabidopsis result from lesions in AtCCD7 and AtCCD8. Both mutants display a dramatic increase in lateral branching and are believed to be impaired in the synthesis of an unidentified compound that inhibits axillary meristem development. To determine the biochemical function of AtCCD7, the protein was expressed in carotenoid-accumulating strains of Escherichia coli. The activity of AtCCD7 was also tested in vitro with several of the most common plant carotenoids. It was shown that the recombinant AtCCD7 protein catalyzes a specific 9-10 cleavage of beta-carotene to produce the 10 black triangle down-apo-beta-carotenal (C27) and beta-ionone (C13). When AtCCD7 and AtCCD8 were co-expressed in a beta-carotene-producing strain of E. coli, the 13-apo-beta-carotenone (C18) was produced. The C18 product appears to result from a secondary cleavage of the AtCCD7-derived C27 product. The sequential cleavages of beta-carotene by AtCCD7 and AtCCD8 are likely the initial steps in the synthesis of a carotenoid-derived signaling molecule that is necessary for the regulation lateral branching.     

Comparison of metal-ion-dependent cleavages of RNA by a DNA enzyme and a hammerhead ribozyme

Joyce's DNA enzyme catalyzes cleavage of RNAs with almost the same efficiency as the hammerhead ribozyme. The cleavage activity of the DNA enzyme was pH dependent, and the logarithm of the cleavage rate increased linearly with pH from pH 6 to pH 9 with a slope of approximately unity. The existence of an apparent solvent isotope effect, with cleavage of RNA by the DNA enzyme in H(2)O being 4.3 times faster than cleavage in D(2)O, was in accord with the interpretation that, at a given pH, the concentration of the active species (deprotonated species) is 4.3 times higher in H(2)O than the concentration in D(2)O. This leads to the intrinsic isotope effect of unity, demonstrating that no proton transfer occurs in the transition state in reactions catalyzed by the DNA enzyme. Addition of La(3+) ions to the Mg(2+)-background reaction mixture inhibited the DNA enzyme-catalyzed reactions, suggesting the replacement of catalytically and/or structurally important Mg(2+) ions by La(3+) ions. Similar kinetic features of DNA enzyme mediated cleavage of RNA and of hammerhead ribozyme-mediated cleavage suggest that a very similar catalytic mechanism is used by the two types of enzyme, despite their different compositions.     

Spectrofluorometric assay of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase activity

A simple, rapid, and sensitive spectrofluorometric assay for 15-hydroxyprostaglandin dehydrogenase activity was developed in which the rate of production of NADH was monitored. The cytosolic fraction prepared from human placental tissue was employed as the enzyme source. The assay was conducted at pH 9.5 since 15-ketoprostaglandin Δ¹³-reductase and NADH oxidase activities were inhibited at this pH, thereby minimizing the interference of the reactions catalyzed by these enzymes in the assay of prostaglandin dehydrogenase activity.     

Purification and characterization of a paired basic residue-specific prohormone-converting enzyme from bovine pituitary neural lobe secretory vesicles

The neuropeptides arginine vasopressin and oxytocin are generated from their prohormones in the hypothalamoneurohypophysial system by enzymatic cleavages at paired basic residues (i.e. Lys-Arg). This study describes the purification of an enzyme from bovine neural lobe secretory vesicles, the putative site of this processing, which is capable of cleaving several prohormones at paired basic residues. The enzyme is a glycoprotein of Mr approximately 70,000 and has an acidic pH maximum. It processes the heterologous precursors pro-opiomelanocortin and insulin at paired basic residues in a manner similar to a pro-opiomelanocortin-converting enzyme derived from bovine intermediate lobe secretory vesicles which has been described previously. In addition, the neural lobe-derived converting enzyme cleaves the human vasopressin prohormone in vitro to yield arginine vasopressin-Gly10-Lys11-Arg12 as the major vasopressin cleavage product. This indicates that the enzymatic cleavage in the vasopressin precursor occurred primarily on the carboxyl side of the arginine in the pair of Lys-Arg basic residues separating the vasopressin peptide from the neurophysin moiety in the precursor. The properties of the neural and intermediate lobe-derived enzymes are virtually identical, raising the possibility that a family of similar enzymes may be responsible for cleaving a number of prohormones at paired basic residues in different tissues.     

The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA

The RF IV form of M13 DNA was synthesized enzymatically in vitro, using the viral (+)strand as template, to contain phosphorothioate-modified internucleotidic linkages of the Rp configuration on the 5' side of every base of a particular type in the newly-synthesized (-)strand. Twenty nine restriction enzymes were then tested for their reactions with the appropriate modified DNA types having a phosphorothioate linkage placed exactly at the cleavage site(s) of these enzymes in the (-)strand. Eleven of the seventeen restriction enzymes tested that had recognition sequences of five bases or more could be used to convert the phosphorothioate DNA entirely into the nicked form, either by simply allowing the reaction to go to completion with excess enzyme (Ava I, Ava II, Ban II, Hind II, Nci I, Pst I or Pvu I) or by stopping the reaction at the appropriate time before the nicked DNA is linearized (Bam HI, Bgl I, Eco RI or Hind III). Only modification of the exact cleavage site in the (-)strand could block linearization by the first class of enzymes. The results presented imply that the restriction enzyme-directed nicking of phosphorothioate M13 DNA occurs exclusively in the (+)strand.     

Sequence changes in both flanking sequences of a pre-tRNA influence the cleavage specificity of RNase P.

The cleavage specificities of the RNase P holoenzymes from Escherichia coli and the yeast Schizosaccharomyces pombe and of the catalytic M1 RNA from E. coli were analyzed in 5'-processing experiments using a yeast serine pre-tRNA with mutations in both flanking sequences. The template DNAs were obtained by enzymatic reactions in vitro and transcribed with phage SP6 or T7 RNA polymerase. The various mutations did not alter the cleavage specificity of the yeast RNase P holoenzyme; cleavage always occurred predominantly at position G + 1, generating the typical seven base-pair acceptor stem. In contrast, the specificity of the prokaryotic RNase P activities, i.e. the catalytic M1 RNA and the RNase P holoenzyme from E. coli, was influenced by some of the mutated pre-tRNA substrates, which resulted in an unusual cleavage pattern, generating extended acceptor stems. The bases G - 1 and C + 73, forming the eighth base pair in these extended acceptor stems, were an important motif in promoting the unusual cleavage pattern. It was found only in some natural pre-tRNAs, including tRNA(SeCys) from E. coli, and tRNAs(His) from bacteria and chloroplasts. Also, the corresponding mature tRNAs in vivo contain an eight base pair acceptor stem. The presence of the CCA sequence at the 3' end of the tRNA moiety is known to enhance the cleavage efficiency with the catalytic M1 RNA. Surprisingly, the presence or absence of this sequence in two of our substrate mutants drastically altered the cleavage specificity of M1 RNA and of the E. coli holoenzyme, respectively. Possible reasons for the different cleavage specificities of the enzymes, the influence of sequence alterations and the importance of stacking forces in the acceptor stems are discussed.     

Isolation and characterization of a dinucleoside triphosphatase from Saccharomyces cerevisiae.

An enzyme able to cleave dinucleoside triphosphates has been purified 3,750-fold from Saccharomyces cerevisiae. Contrary to the enzymes previously shown to catabolize Ap4A in yeast, this enzyme is a hydrolase rather than a phosphorylase. The dinucleoside triphosphatase molecular ratio estimated by gel filtration is 55,000. Dinucleoside triphosphatase activity is strongly stimulated by the presence of divalent cations. Mn2+ displays the strongest stimulating effect, followed by Mg2+, Co2+, Cd2+, and Ca2+. The Km value for Ap3A is 5.4 microM (50 mM Tris-HCl [pH 7.8], 5 mM MgCl2, and 0.1 mM EDTA; 37 degrees C). Dinucleoside polyphosphates are substrates of this enzyme, provided that they contain more than two phosphates and that at least one of the two bases is a purine (Ap3A, Ap3G, Ap3C, Gp3G, Gp3C, m7Gp3A, m7Gp3G, Ap4A, Ap4G, Ap4C, Ap4U, Gp4G, and Ap5A are substrates; AMP, ADP, ATP, Ap2A, and Cp4U are not). Among the products, a nucleoside monophosphate is always formed. The specificity of cleavage of methylated dinucleoside triphosphates and the molecular weight of dinucleoside triphosphatase indicate that this enzyme is different from the mRNA decapping enzyme previously characterized (A. Stevens, Mol. Cell. Biol. 8:2005-2010, 1988).     

Mechanistic studies on beta-ketoacyl thiolase from Zoogloea ramigera: identification of the active-site nucleophile as Cys89, its mutation to Ser89, and kinetic and thermodynamic characterization of wild-type and mutant enzymes

Thiolase proceeds via covalent catalysis involving an acetyl-S-enzyme. The active-site thiol nucleophile is identified as Cys89 by acetylation with [14C]acetyl-CoA, rapid denaturation, tryptic digestion, and sequencing of the labeled peptide. The native acetyl enzyme is labile to hydrolytic decomposition with t 1/2 of 2 min at pH 7, 25 degrees C. Cys89 has been converted to the alternate nucleophile Ser89 by mutagenesis and the C89S enzyme overproduced, purified, and assessed for activity. The Ser89 enzyme retains 1% of the Vmax of the Cys89 enzyme in the direction of acetoacetyl-CoA thiolytic cleavage and 0.05% of the Vmax in the condensation of two acetyl-CoA molecules. A covalent acetyl-O-enzyme intermediate is detected on incubation with [14C]acetyl-CoA and isolation of the labeled Ser89-containing tryptic peptide. Comparisons of the Cys89 and Ser89 enzymes have been made for kinetic and thermodynamic stability of the acetyl enzyme intermediates both by isolation and by analysis of [32P]CoASH/acetyl-CoA partial reactions and for rate-limiting steps in catalysis with trideuterioacetyl-CoA.     

Substrate activity of synthetic formyl phosphate in the reaction catalyzed by formyltetrahydrofolate synthetase

Formyl phosphate, a putative enzyme-bound intermediate in the reaction catalyzed by formyltetrahydrofolate synthetase (EC 6.3.4.3), was synthesized from formyl fluoride and inorganic phosphate [Jaenicke, L. v., & Koch, J. (1963) Justus Liebigs Ann. Chem. 663, 50-58], and the product was characterized by 31P, 1H, and 13C nuclear magnetic resonance (NMR). Measurement of hydrolysis rates by 31P NMR indicates that formyl phosphate is particularly labile, with a half-life of 48 min in a buffered neutral solution at 20 degrees C. At pH 7, hydrolysis occurs with P-O bond cleavage, as demonstrated by 18O incorporation from H2(18)O into Pi, while at pH 1 and pH 13 hydrolysis occurs with C-O bond cleavage. The substrate activity of formyl phosphate was tested in the reaction catalyzed by formyltetrahydrofolate synthetase isolated from Clostridium cylindrosporum. Formyl phosphate supports the reaction in both the forward and reverse directions. Thus, N10-formyltetrahydrofolate is produced from tetrahydrofolate and formyl phosphate in a reaction mixture that contains enzyme, Mg(II), and ADP, and ATP is produced from formyl phosphate and ADP with enzyme, Mg(II), and tetrahydrofolate present. The requirements for ADP and for tetrahydrofolate as cofactors in these reactions are consistent with previous steady-state kinetic and isotope exchange studies, which demonstrated that all substrate subsites must be occupied prior to catalysis. The k cat values for both the forward and reverse directions, with formyl phosphate as the substrate, are much lower than those for the normal forward and reverse reactions. Kinetic analysis of the formyl phosphate supported reactions indicates that the low steady-state rates observed for the synthetic intermediate are most likely due to the sequential nature of the normal reaction.     

Isolation and characterization of a thermostable intracellular enzyme with peroxidase activity from Bacillus sphaericus

During a screening for bacteria producing enzymes with peroxidase activity, a Bacillus sphaericus strain was isolated. This strain was found to contain an intracellular enzyme with peroxidase activity. The native enzyme had a molecular mass of above 300 kDa and precipitated at a salt concentration higher than 0.1 M. Proteolytic digestion with trypsin reduced the molecular mass of the active enzyme to 13 kDa (dimer) or 26 kDa (tetramer) and increased its solubility, allowing purification to homogeneity. Spectroscopic investigations showed the enzyme to be a hemoenzyme containing heme c as the covalently bound prosthetic group. The enzyme was stable up to 90 degrees C and at alkaline conditions up to pH 11, with a pH optimum at pH 8.5. It could be visualized by activity staining after SDS-PAGE and showed activity with a number of typical substrates for peroxidases, e.g., 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) diammonium salt, guaiacol and 2,4-dichlorophenol; however the enzyme had no catalase and cytochrome c peroxidase activity.     

Urea amidolyase of Candida utilis. Characterization of the urea cleavage reactions.

Evidence is presented that the enzymes catalyzing the three reactions involved in urea cleavage in Candida utilis, biotin carboxylation, urea carboxylation, and allophanate hydrolysis occur as a complex of enzymes. The allophanate-hydrolyzing activity could not be separated from the urea-cleaving activity using common methods of protein purification. Further, urea cleavage and allophanate hydrolysis activities are induced coordinately in cells grown on various nitrogen sources. The reactions involved in urea cleavage can be distinguished from one another on the basis of their sensitivities to (a) heat, (b) pH, and (c) chemical inhibitors. Evidence is presented for the product of the first reaction in urea cleavage, biotin carboxylation. Production of carboxylated enzyme is ATP dependent and avidin sensitive. Carboxylated enzyme is not observed in the presence of 1 mM urea.