Inhibition in vitro of lipogenic enzymes from bovine (Bos taurus) mammary tissue by methylmalonyl-coenzyme A and coenzyme A

Bovine mammary fatty acid synthetase was inhibited by approximately 50% by 40 microM methylmalonyl-CoA; this inhibition was competitive with respect to malonyl-CoA (apparent Ki = 11 microM). Similarly, 6.25 microM coenzyme A inhibited the synthetase by 35% and this inhibition was again competitive (apparent Ki = 1.7 microM). Apparent Km for malonyl-CoA was 29 microM. The short-chain dicarboxylic acids malonic, methylmalonic and ethylmalonic at high concentrations (160-320 microM) and ATP (5 mM) enhanced the synthetase activity by about 50% respectively; the activating effects of methylmalonic acid and ATP on the synthetase were additive. Methylmalonyl-CoA at 50 microM concentration inhibited the partially purified acetyl-CoA carboxylase uncompetitively by 10% and the propionyl-CoA carboxylase activity of the enzyme preparation competitively (apparent Ki = 21 microM) by 40%. Malonyl-CoA also inhibited the acetyl-CoA carboxylase activity competitively (apparent Ki = 7 microM) by 35% and the propionyl-CoA carboxylating activity of the preparation competitively (apparent Ki = 4 microM) by 82%. The possibility that methylmalonyl-CoA may be a causal factor in the aetiology of the low milk-fat syndrome in high yielding dairy cows is discussed.     

Human liver methenyltetrahydrofolate synthetase: improved purification and increased affinity for folate polyglutamate substrates

Methenyltetrahydrofolate synthetase (5-formyltetrahydrofolate cyclodehydrase (cyclo-ligase) (ADP-forming) EC 6.3.3.2) catalyzes the ATP- and Mg2+-dependent transformation of 5-formyltetrahydrofolate (leucovorin) to 5,10-methenyltetrahydrofolate. The enzyme has been purified 49,000-fold from human liver by a two-column procedure with Blue Sepharose followed by folinate-Sepharose chromatography. It appears as a single band both on SDS-polyacrylamide gel electrophoresis (Mr 27,000) and on isoelectric focusing (pI = 7.0) and is monomeric, with a molecular weight of 27,000 on gel filtration. Initial-velocity studies suggest that the enzyme catalyzes a sequential mechanism and at 30 degrees C and pH 6.0 the turnover number is 1000 min-1. The enzyme has a higher affinity for its pentaglutamate substrate (Km = 0.6 microM) than for the monoglutamate (Km = 2 microM). The antifolate methotrexate has no inhibitory effect at concentrations up to 350 microM, while methotrexate pentaglutamate is a competitive inhibitor with a Ki = 15 microM. Similarly, dihydrofolate monoglutamate is a weak inhibitor with a Ki = 50 microM, while the pentaglutamate is a potent competitive inhibitor with a Ki of 3.8 microM. Thus, dihydrofolate and methotrexate pentaglutamates could regulate enzyme activity and help explain why leucovorin fails to rescue cells from high concentrations of methotrexate.     

Inhibition of acetoacetyl-CoA synthetase from rat liver by fatty acyl-CoAs

The activity of acetoacetyl-CoA synthetase from rat liver was found to be negatively regulated by coenzyme A, fatty acyl-CoAs and acetoacetyl-CoA in vitro. With increasing concentrations of coenzyme A (substrate inhibition occurring at concentrations higher than 50 microM) the pH optimum shifted toward the acidic side (7.5-8.5 with 5 microM coenzyme A and 6.5-7.0 with 500 microM coenzyme A), in parallel with progressively decreasing enzyme activity. Fatty acyl-CoAs of various chain lengths dose-dependently inhibited acetoacetyl-CoA synthetase from rat liver, but much less effectively a similar enzyme from a bacterium, Zoogloea ramigera I-16-M. Palmitoyl-CoA, the most potent inhibitor of the rat liver enzyme, with an apparent Ki value of 9.8 microM, apparently inhibited the enzyme below its critical micellar concentration, not due to its detergent action. Acetoacetyl-CoA showed product inhibition with a Ki value of 15 microM. These results suggest a possible physiological regulation mechanism for this enzyme with respect to fatty acid biosynthesis.     

Polyamine synthesis in mammalian tissues. Isolation and characterization of spermine synthase from bovine brain

Spermine synthase, a propylamine transferase, which catalyses the biosynthesis of spermine from S-methyladenosylhomocystemine and spermidine has been purified to an apparent homogeneity (about 6000-fold) from bovine brain using spermine-Sepharose affinity chromatography. The enzyme preparation was free from S-adenosylmethionine decarboxylase and spermidine synthase activities. The molecular Stokes radius of the enzyme was calculated to be 4.16 nm. The enzyme has an apparent molecular weight of approximately 88 000, composing of two subunits of equal size. The enzyme showed a broad pH optimum between 7.0 and 8.0 and an acidic isoelectric point at pH 5.10. The apparent Km values for S-methyladenosylhomocysteamine was 0.6 microM and about 60 microM for spermidine. The enzyme showed strict specificity to spermidine as the propylamine acceptor. Both the reaction products, spermine and 5'-methylthioadenosine inhibited the enzyme activity, methylthioadenosine being a powerful competitive inhibitor with respect to S-methyladenosylhomocysteamine (Ki value of about 0.3 microM). Putrescine also inhibited competitively with respect to spermidine (Ki value of about 1.7 mM). Spermine synthase had no requirements for metal or other cofactors.     

Adenylosuccinate synthetase from Dictyostelium discoideum: effects of hadacidin analogs and binding of [14C]hadacidin

The enzyme adenylosuccinate (sAMP) synthetase has been partially purified from Dictyostelium discoideum using hadacidin-Sepharose 4B affinity chromatography, anion-exchange high-performance liquid chromatography (HPLC), and gel-filtration HPLC, resulting in a 2600-fold purification. Using a newly developed HPLC procedure to assay activity, it has been found that D. discoideum adenylosuccinate synthetase activity has apparent Km values for the substrates IMP, GTP, and aspartate of 36, 23, and 714 microM, respectively. The analog guanosine-5'-(beta, gamma-imino)triphosphate was found to be an inhibitor of GTP with a Ki of 15 microM, and IMP was competitively inhibited by its analog formycin B monophosphate with a Ki of 80 microM. An analysis of these kinetic data showed a pattern consistent with a fully random terter mechanism. Hadacidin, an analog of aspartate, was an inhibitor of that substrate at 86 microM. Other analogs of hadacidin were synthesized and examined for their effect on the sAMP synthetase activity. Compared to hadacidin, which produced 100% inhibition at 5 mM, it was observed that N-acetyl-N-hydroxyglycine, N-formylglycine, N-acetylglycine, and N-hydroxyglycine all inhibited between 50 and 75%, with N-(thiocarboxy)-L-aspartic anhydride less effective at 27%, and N-benzoylglycine at only 6%. N-Formylsarcosine, N-acetylmethionine, O-methylpyruvate oxime, and hadacidin methylester had no effect at this concentration. The adenylosuccinate synthetase activity was dependent on metal ions with maximum activity being obtained with Mg2+. The ability of the aspartate analog hadacidin to bind to the purified adenylosuccinate synthetase was demonstrated using anion-exchange HPLC and [formyl-14C]hadacidin. The radioactivity coeluted with the adenylosuccinate synthetase and the bound, radiolabeled hadacidin was displaced by excess aspartate.     

Methenyltetrahydrofolate synthetase prevents the inhibition of phosphoribosyl 5-aminoimidazole 4-carboxamide ribonucleotide formyltransferase by 5-formyltetrahydrofolate polyglutamates

Methenyltetrahydrofolate synthetase (EC 6.3.3.2) catalyzes the irreversible ATP and Mg2+-dependent transformation of 5-formyltetrahydrofolate (N5-HCO-H4-pteroylglutamic acid (PteGlu] to 5,10-methenyltetrahydrofolate. The physiological function of this reaction remains unknown even though it is potentially involved in the intracellular metabolism of the large doses of N5-HCO-H4-PteGlu (leucovorin) administered to cancer patients. We have tried to elucidate methenyltetrahydrofolate synthetase's physiological role by examining the consequences of its inhibition in MCF-7 human breast cancer cells by the folate analog 5-formyltetrahydrohomofolate (fTHHF), a potent competitive inhibitor with a Ki of 1.4 microM. fTHHF inhibited MCF-7 cell growth with an IC50 of 2.0 microM during 72-h exposures, and this effect was fully reversible by hypoxanthine but not thymidine, indicating specific inhibition of de novo purine synthesis. A correlation was observed between increases in intracellular N5-HCO-H4-PteGlu concentrations following fTHHF and cell growth inhibition. De novo purine synthesis was inhibited at the second folate-dependent enzyme, phosphoribosyl aminoimidazole-carboxamide formyltransferase (AICAR transferase; EC 2.1.2.3), as determined by aminoimidazole carboxamide rescue and azaserine inhibition studies. N5-HCO-H4-PteGlu pentaglutamate was a potent inhibitor of purified MCF-7 cell AICAR transferase with a Ki of 3.0 microM while the monoglutamate was not an inhibitor up to 10 microM and fTHHF was only weakly inhibitory with a Ki of 16 microM. These findings suggest that methenyltetrahydrofolate synthetase activity is needed to prevent de novo purine synthesis inhibition by N5-HCO-H4-PteGlu polyglutamates.     

Cyclopentenylcytosine triphosphate. Formation and inhibition of CTP synthetase

Cyclopentenylcytosine (CPEC) is phosphorylated in L1210 cells with CPEC triphosphate as the major metabolite. Partially purified uridine-cytidine kinase catalyzes the initial phosphorylation of cyclopentenylcytosine with an apparent Km of 196 +/- 9 microM, and cyclopentenylcytosine is a competitive inhibitor of cytidine phosphorylation by this enzyme with a Ki value of 144 +/- 14 microM. Examination of the CTP synthetase activity in extracts of L1210 cells revealed a dose-dependent decrease on exposure of cells to CPEC. Synthesis of CPEC triphosphate by an enzymatic method permitted direct examination of the inhibition of partially purified CTP synthetase. CPEC triphosphate inhibited bovine CTP synthetase with a median inhibitory concentration of 6 microM, whereas CPEC mono- and diphosphates were ineffective. CTP synthetase showed a classical Michaelis-Menten hyperbolic plot of velocity and UTP concentration in the presence of saturating concentrations of ATP and glutamine, but CPEC triphosphate induced sigmoidal kinetic plots. The Hill coefficient was calculated to be 3.2.     

The isolation of a peptide from the catalytic domain of Bacillus stearothermophilus tryptophyl-tRNA synthetase. The interaction of Brown MX-5BR with tyrosyl-tRNA synthetase.

Tryptophyl-tRNA synthetase is irreversibly inactivated by Procion Brown MX-5BR with an apparent dissociation constant (KD) of 8.8 microM and maximum rate of inactivation k3 0.192 s-1. The specificity of the interaction is supported by two previously reported observations. Firstly, Brown MX-5BR inactivation of tryptophyl-tRNA synthetase is inhibited by substrates, and secondly, the animated derivative of Brown MX-5BR is a competitive inhibitor of tryptophyl-tRNA synthetase with a Ki of 2 X 10(-4) M with respect to both tryptophan and ATP. Tryptic digestion of the dye-affinity-labelled enzyme and subsequent resolution of the peptides by h.p.l.c. yielded one major dye-peptide peak. Amino acid sequence analysis resulted in the identification of the dye-binding domain centred on lysine-178. Tyrosyl-tRNA synthetase is also inactivated by Procion Brown MX-5BR, and this inactivation is prevented by ATP but not by tyrosine. The interaction of tyrosyl-tRNA synthetase with hydroxylated Brown MX-5BR exhibited non-competitive kinetics with respect to the amino acid-binding site and competitive kinetics against ATP with a Ki of 6 X 10(-6) M.     

Pseudomonas aeruginosa possesses two novel regulatory isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

In Pseudomonas aeruginosa the initial enzyme of aromatic amino acid biosynthesis, 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase, has been known to be subject to feedback inhibition by a metabolite in each of the three major pathway branchlets. Thus, an apparent balanced multieffector control is mediated by L-tyrosine, by L-tryptophan, and phenylpyruvate. We have now resolved DAHP synthase into two distinctive regulatory isozymes, herein denoted DAHP synthase-tyr (Mr = 137,000) and DAHP synthase-trp (Mr = 175,000). DAHP synthase-tyr comprises greater than 90% of the total activity. L-Tyrosine was found to be a potent effector, inhibiting competitively with respect to both phosphoenolpyruvate (Ki = 23 microM) and erythrose 4-phosphate (Ki = 23 microM). Phenylpyruvate was a less effective competitive inhibitor: phosphoenolpyruvate (Ki = 2.55 mM) and erythrose 4-phosphate (Ki = 1.35 mM). DAHP synthase-trp was found to be inhibited noncompetitively by L-tryptophan with respect to phosphoenolpyruvate (Ki = 40 microM) and competitively with respect to erythrose 4-phosphate (Ki = 5 microM). Chorismate was a relatively weak competitive inhibitor: phosphoenolpyruvate (Ki = 1.35 mM) and erythrose 4-phosphate (Ki = 2.25 mM). Thus, each isozyme is strongly inhibited by an amino acid end product and weakly inhibited by an intermediary metabolite.     

Interaction of pseudomonic acid A with Escherichia coli B isoleucyl-tRNA synthetase.

Sodium pseudomonate was shown to be a powerful competitive inhibitor of Escherichia coli B isoleucyl-tRNA synthetase (Ile-tRNA synthetase). The antibiotic competitively inhibits (Ki 6 nM; cf. Km 6.3 microM), with respect top isoleucine, the formation of the enzyme . Ile approximately AMP complex as measured by the pyrophosphate-exchange reaction, and has no effect on the transfer of [14C]isoleucine from the enzyme . [14C]Ile approximately AMP complex to tRNAIle. The inhibitory constant for the pyrophosphate-exchange reaction was of the same order as that determined for the inhibition of the overall aminoacylation reaction (Ki 2.5 nM; cf. Km 11.1 microM). Sodium [9'-3H]pseudomonate forms a stable complex with Ile-tRNA synthetase. Gel-filtration and gel-electrophoresis studies showed that the antibiotic is only fully released from the complex by 5 M-urea treatment or boiling in 0.1% sodium dodecyl sulphate. The molar binding ratio of sodium [9'-3H]pseudomonate to Ile-tRNA synthetase was found to be 0.85:1 by equilibrium dialysis. Aminoacylation of yeast tRNAIle by rat liver Ile-tRNA synthetase was also competitively inhibited with respect to isoleucine, Ki 20 microM (cf. Km 5.4 microM). The Km values for the rat liver and E. coli B enzymes were of the same order, but the Ki for the rat liver enzyme was 8000 times the Ki for the E. coli B enzyme. This presumably explains the low toxicity of the antibiotic in mammals.     

Synthesis of glutaminyl adenylate analogues that are inhibitors of glutaminyl-tRNA synthetase

Glutaminol adenylate 5 is a competitive inhibitor of glutaminyl-tRNA synthetase with respect to glutamine (Ki = 280 nM) and to ATP (Ki = 860 nM). The corresponding methyl phosphate ester 4 is a weaker inhibitor (Ki approximately 10 microM) with respect to glutamine.     

Effects of acyclovir and its metabolites on purine nucleoside phosphorylase

Acyclovir (9-(2-hydroxyethoxymethyl)guanine), the clinically useful antiherpetic agent, is an "acyclic" analogue of 2'-deoxyguanosine. Purine nucleoside phosphorylase partially purified from human erythrocytes did not catalyze detectable phosphorolysis of this drug or any of its metabolites (less than 0.07% of the rate with Guo). However, these compounds were competitive inhibitors of this enzyme with Ino as the variable substrate. Acyclovir per se was a relatively weak inhibitor. Its Ki value (91 microM) was much greater than that for its 8-hydroxy metabolite (Ki = 4.7 microM) but less than that for its carboxylic acid metabolite (9-carboxymethoxy-methylguanine) (K'i = 960 microM). The phosphorylated metabolites of acyclovir were more potent inhibitors than were their guanine nucleotide counterparts. At a phosphate concentration of 50 mM, the apparent Ki values for the mono- (120 microM), di- (0.51 microM), and tri (43 microM)-phosphate esters of acyclovir were 1/2, 1/1200, and 1/26 those for dGMP, dGDP, and dGTP, respectively. The concentration of phosphate did not markedly affect the Ki value of acyclovir but dramatically affected those of its phosphorylated metabolites and their nucleotide counterparts. Decreasing phosphate to a physiological concentration (1 mM) decreased the apparent Ki values for the mono-, di-, and triphosphate esters of acyclovir to 6.6, 0.0087, and 0.31 microM, respectively. Inhibition of the enzyme by acyclovir diphosphate was also influenced by pH. This metabolite of acyclovir is the most potent inhibitor of purine nucleoside phosphorylase reported to date. It has some features of a "multisubstrate" analogue inhibitor.     

Solubilisation and reconstitution of acylcoenzyme A:estradiol-17 beta acyltransferase

Acylcoenzyme A:estradiol-17 beta acyltransferase in microsomes of bovine placenta cotyledons was strongly membrane bound. The enzyme was solubilised from microsomes by sodium cholate and was reconstituted into phospholipid vesicles. The apparent Km for estradiol-17 beta was 11 microM which was close to the value of 8 microM previously found with the membrane-bound enzyme. Testosterone was also a substrate for the reconstituted enzyme (apparent Km 62 microM) and was a competitive inhibitor (Ki 74 microM) of the acylation of estradiol-17 beta. Although various long-chained fatty acyl CoAs acted as acyl donors, these proved to have widely differing apparent Km values with palmitoleoyl CoA having the highest affinity (Km 24 microM) and arachidonoyl CoA the lowest affinity (Km 330 microM).     

Purification and characterization of adenine phosphoribosyltransferase from mouse mammary carcinoma FM3A cells in culture

Adenine phosphoribosyltransferase has been purified to apparent homogeneity from mouse mammary tumor FM3A cells. The purified enzyme, with a specific activity of 20.6 X 10(6) units/g protein at 30 degrees C, was homogeneous as judged by polyacrylamide gel electrophoresis and Ouchterlony double immunodiffusion analysis. The native enzyme had a molecular weight of 44,000 and a subunit composition of 23,000. Apparent Km values for adenine and 5-phosphoribosyl-1-pyrophosphate (PRib-PP) were 6.6 microM and 1.2 microM, respectively. Free Mg2+ was an essential activator with a half-maximal effect at 0.4 mM. AMP was an inhibitor, competitive with PRib-PP, and the Ki value was estimated to be 24 microM. The enzyme activity was not significantly affected by 2,6-diaminopurine, 4-carbamoylimidazolium 5-olate, 8-azaadenine, and 2-fluoro-6-aminopurine. An antibody against the purified mouse adenine phosphoribosyltransferase was raised in a rabbit. The enzyme derived from either mouse, Chinese hamster, or human cells was completely neutralized and precipitated by this antibody, indicating that these enzymes share a common antigenic determinant.     

Regulation of deoxyadenosine and nucleoside analog phosphorylation by human placental adenosine kinase

The enzymes responsible for the phosphorylation of deoxyadenosine and nucleoside analogs are important in the pathogenesis of adenosine deaminase deficiency and in the activation of specific anticancer and antiviral drugs. We examined the role of adenosine kinase in catalyzing these reactions using an enzyme purified 4000-fold (2.1 mumol/min/mg) from human placenta. The Km values of deoxyadenosine and ATP are 135 and 4 microM, respectively. Potassium and magnesium are absolute requirements for deoxyadenosine phosphorylation, and 150 mM potassium and 5 mM MgCl2 are critical for linear kinetics. With only 0.4 mM MgCl2 in excess of ATP levels, the Km for deoxyadenosine is increased 10-fold. ADP is a competitive inhibitor with a Ki of 13 microM with variable MgATP2-, while it is a mixed inhibitor with a Ki and Ki' of 600 and 92 microM, respectively, when deoxyadenosine is variable. AMP is a mixed inhibitor with Ki and Ki' of 177 and 15 microM, respectively, with variable deoxyadenosine; it is a non-competitive inhibitor with a Ki of 17 microM and Ki' of 27 microM with variable ATP. Adenosine kinase phosphorylates adenine arabinoside with an apparent Km of 1 mM using deoxyadenosine kinase assay conditions. The Km values for 6-methylmercaptopurine riboside and 5-iodotubercidin, substrates for adenosine kinase, are estimated to be 4.5 microM and 2.6 nM, respectively. Other nucleoside analogs are potent inhibitors of deoxyadenosine phosphorylation, but their status as substrates remains unknown. These data indicate that deoxyadenosine phosphorylation by adenosine kinase is primarily regulated by its Km and the concentrations of Mg2+, ADP, and AMP. The high Km values for phosphorylation of deoxyadenosine and adenine arabinoside suggest that adenosine kinase may be less likely to phosphorylate these nucleosides in vivo than other enzymes with lower Km values. Adenosine kinase appears to be important for adenosine analog phosphorylation where the Michaelis constant is in the low micromolar range.     

Purification and characterization of 3-deoxy-D-manno-octulosonate 8-phosphate synthetase from Escherichia coli

3-Deoxy-D-manno-octulosonate (KDO)-8-phosphate synthetase has been purified 450-fold from frozen Escherichia coli B cells. The purified enzyme catalyzed the stoichiometric formation of KDO-8-phosphate and Pi from phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate. The enzyme showed no metal requirement for activity and was inhibited by 1 mM Cd2+, Cu2+, Zn2+, and Hg2+. The inhibition by Hg2+ could be reversed by dithiothreitol. The optimum temperature for enzyme activity was determined to be 45 degrees C, and the energy of activation calculated by the Arrhenius equation was 15,000 calories (ca. 3,585 J) per mol. The enzyme activity was shown to be pH and buffer dependent, showing two pH optima, one at pH 4.0 to 6.0 in succinate buffer and one at pH 9.0 in glycine buffer. The isoelectric point of the enzyme was 5.1. KDO-8-phosphate synthetase had a molecular weight of 90,000 +/- 6,000 as determined by molecular sieving through G-200 Sephadex and by Ferguson analysis using polyacrylamide gels. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 90,000-molecular-weight native enzyme was composed of three identical subunits, each with an apparent molecular weight of 32,000 +/- 4,000. The enzyme had an apparent Km for D-arabinose-5-phosphate of 2 X 10(-5) M and an apparent Km for PEP of 6 X 10(-6) M. No other sugar or sugar-phosphate could substitute for D-arabinose-5-phosphate. D-Ribose-5-phosphate was a competitive inhibitor of D-arabinose-5-phosphate, with an apparent Ki of 1 X 10(-3) M. The purified enzyme has been utilized to synthesize millimole quantities of pure KDO-8-phosphate.     

Competitive inhibitors of rabbit hepatic microsomal steroid 12 alpha-hydroxylase

The sterols 7 alpha-hydroxycholest-4-en-3-one (I) and 5 alpha-cholestane-3 alpha,7 alpha-diol (II) are competitive inhibitors for rabbit hepatic microsomal preparations of steroid 12 alpha-hydroxylase with apparent Ki values of 56 and 93 microM, respectively. To ascertain the optimum structure for a substrate with maximal enzymic activity, nine sterols or steroidal acids containing the 7 alpha-hydroxy-4-en-3-one or 3 alpha,7 alpha-dihydroxy-5 alpha configuration were prepared and studied as inhibitors with enzyme preparations in the presence of NADPH, oxygen and appropriate cofactors. Although each of these compounds exhibited competitive inhibition, the best inhibitor for sterol (I) was 7 alpha,25-dihydroxycholest-4-en-3-one (IV) (Ki 36 microM). Steroidal acids (3-oxo-7 alpha-hydroxychol-4-enoic acid and 3-oxo-7 alpha-hydroxy-4-cholene-24-carboxylic acid) were poor inhibitors (Ki 1080 and 654 microM, respectively). For sterol (II) the best inhibitors were sterol (IV) (Ki 35 microM) and 5 alpha-cholestane-3 alpha,7 alpha,25-triol (VIII) (Ki 45 microM). The 12 alpha-hydroxylated products of sterols (I) and (IV) were less tightly bound to the enzyme (Ki 88 and 98 microM, respectively) in the presence of sterol (II). Allochenodeoxycholic acid (Ki 495 microM) was not a good inhibitor for sterol (II). 12 alpha-Hydroxylated products of sterols (IV) and (VIII) were isolated from larger scale incubations, separated by HPLC and identified by mass spectrometry.     

Isolation and properties of phosphoribosyl-aminoimidazole-succinocarboxyamide-synthestase from Saccharomyces cerevisiae yeasts

An isolation procedure for phosphoribosyl succinocarboxamideaminoimidazole synthetase (SAICAR synthetase) (EC 6.3.2.6) has been developed. Pure SAICAR synthetase was found to be a monomeric protein with the apparent molecular weight of 36 kDa. The Michaelis constant for the three substrates of the reaction are 1.6 microM for CAIR, 14 microM for ATP and 960 microM for aspartic acid. The structural analogs of CAIR, 5-aminoimidazole ribotide and 5-aminoimidazole-4-carboxamide ribotide, act as competitive inhibitors of SAICAR synthetase. GTP and 2'-dATP can substitute for ATP in the reaction, while CTP and UTP inhibit the enzyme. No structural analogs of the aspartic acid were found to have affinity for SAICAR synthetase. The optimal reaction conditions for the enzyme were established to be at pH 8.0 and magnesium chloride concentration around 5 mM.     

A bisubstrate analog inhibitor for alpha(1----2)-fucosyltransferase

Porcine submaxillary beta-galactoside alpha(1----2)-fucosyltransferase is known to transfer a fucosyl residue from guanosine 5'-diphosphofucose (GDP-fucose) to the 2-OH group of beta-D-galactopyranosides with inversion of configuration at the fucopyranosyl anomeric carbon. A bisubstrate analog (1) of the postulated transition-state for this reaction, which has O-2 of phenyl beta-D-galactopyranoside attached to the terminal phosphorous of GDP through a flexible ethylene bridge, has been chemically synthesized and evaluated as an inhibitor of this enzyme. Compound 1 was found to be a competitive inhibitor with respect to both GDP-fucose and phenyl beta-D-galactopyranoside for both the membrane-bound and soluble forms of the fucosyltransferase. It was also a competitive inhibitor with respect to the alternate acceptor beta DGal(1----3)beta DGlcNAcO(CH2)8-COOMe. The Ki values were in the range 2.3-16 microM. Compound 1 is the first example of a bisubstrate analog inhibitor for a glycosyltransferase which binds to both the acceptor and donor recognition sites of the enzyme. The potential of a bisubstrate analog strategy for the production of specific glycosyltransferase inhibitors is discussed.