Invertase storage stability and sucrose hydrolysis in solids as affected by water activity and glass transition.

Research continues to differentiate the impact of water activity (a(W)) and the glass transition temperature (T(g)) on chemical reactions. Invertase with and without sucrose was incorporated into low and high molecular weight poly(vinylpyrrolidone) model systems (PVP-LMW and PVP-K30, respectively). Invertase activity and sucrose hydrolysis were monitored during storage at a(W) = 0.32-0.75 and 30 degrees C. Pseudo-first-order rate constants for activity loss in PVP-K30 were not different, regardless of the system being glassy or rubbery. In PVP-LMW, invertase stability decreased with increasing a(W). An a(W) > 0.62 was required for sucrose hydrolysis to occur in PVP-LMW. PVP molecular weight appeared to affect invertase stability and reactivity. No dramatic change around T(g) was found in either invertase stability or sucrose hydrolysis, suggesting that T(g)-dictated mobility has a minimal effect on these reactions in amorphous solids.

Ligand-induced conformational changes of thymidylate synthase detected by limited proteolysis.

Limited tryptic proteolysis was used to investigate conformational changes of thymidylate synthase from Lactobacillus casei induced by ligand binding. Most of the identified sites of proteolysis were between R72 and R178, a region that includes a large loop containing residues 90-139 that is absent in thymidylate synthase from most other sources. Hydrolysis at both ends of this region was affected by the presence of dUMP. With dUMP, the preference of initial hydrolysis at the N-terminus of this region was switched from R78 to R72, and hydrolysis at R178 was retarded; the latter effect may be primarily a consequence of steric hinderance since R178 is involved in binding the phosphate moiety of dUMP. Orthophosphate had an effect similar to that of dUMP, not only in retarding hydrolysis at the phosphate binding site (R178) but also in retarding hydrolysis at R78 in favor of R72. Alkylation of the catalytically essential sulfhydryl group of thymidylate synthase by iodoacetamide also resulted in R72 being favored over R78 as a site of initial proteolysis. Its effect on hydrolysis at R178 was, as expected, less than that of dUMP or phosphate. These results indicate that dUMP binding induces conformational changes in thymidylate synthase. Phosphate binding and sulfhydryl alkylation also induce conformational changes similar to those resulting from dUMP binding. While the similarity of the proteolytic behavior of thymidylate synthase in the presence of dUMP or phosphate agrees with the report by Finer-Moore et al.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of pyridoxal phosphate with thymidylate synthase: spectral and equilibrium dialysis studies.

1. Changes in the spectrum of pyridoxal phosphate (PLP) were produced by adding an equimolar amount of native thymidylate synthase, but not by adding denatured enzyme or enzyme modified by sulfhydryl-blocking reagents. 2. The dissociation constant of the thymidylate synthase-PLP complex determined by equilibrium dialysis was 9 +/- 1.6 microM, the maximum number of PLP molecules bound per molecule of native thymidylate synthase was 2.5 +/- 0.4, and the Hill coefficient was 0.97. 3. No evidence of PLP binding was found with denatured thymidylate synthase, and only slight binding was observed when enzyme SH groups were blocked or when the active site was blocked with 5-fluorodeoxyuridylate (FdUMP) and methylene tetrahydrofolate. 4. The presence of dUMP, dTMP, or FdUMP interfered with the binding of PLP to thymidylate synthase, and the presence of equimolar amounts of PLP interfered with the binding of dUMP.

Enzyme selectivity analyses of arylsulfonylamino acid aldose reductase inhibitors.

Arylsulfonylamino acids, displaying a wide range of inhibitory activities versus rat lens aldose reductase (RLAR), were analyzed for enzyme selectivity in several test systems. These RLAR inhibitors were found not to produce significant inhibition of genetically-linked reductases (aldehyde reductase, ALR), catalytically similar reductases (Pachysolen tannophilus xylose reductase, PTXR), functionally distinct oxidoreductases (glutathione reductase, GR, lactate dehydrogenase, LDH, and gamma-transaminase, GABA-T), and thymidylate synthase (TS). These data suggest that aldose reductase differs significantly from other oxidoreductases in its inhibitor binding domain(s). Furthermore, the aldose reductase selectivity demonstrated by the arylsulfonylamino acids suggests that these compounds may not inhibit other key metabolic transformations in various cell types and that they may function as selective probes for studies of the relationship between aldose reductase mediated biochemical changes and the pathologies of chronic diabetes.

1-Phenyl-3-trimethylaminopropyl carbodiimide: a new inhibitor of thymidylate synthase.

Thymidylate synthase (EC 2.1.1.45) from methotrexate-resistant Lactobacillus casei was inactivated by 1-phenyl-3-trimethylaminopropyl carbodiimide (PTC), 1-phenyl-3-dimethyl aminopropyl carbodiimide (PDC), and 1-ethyl-3-dimethyl aminopropyl carbodiimide (EDC). In the presence of excess PTC, the inactivation followed pseudo-first order kinetics; the second order rate constant was approximately 200 M-1min-1 at 30 degrees C. The rate of inactivation by PTC was faster than that by either PDC or EDC. Concentrations of the substrate dUMP greater than 0.15 mM, or of the product dTMP greater than 1.6 mM completely protected the enzyme from inactivation by PTC, but 10 mM dUrd provided very little protection. The rate of inactivation of EDC was reduced by only 40% in the presence of 50 mM dUMP. Nucleophiles (sulfanilic acid, glycine methyl ester, or glycine ethyl ester) had no effect on the rate of inactivation by PTC. The complete inactivation of thymidylate synthase by PTC was accompanied by the incorporation of approximately 2 mols of 14C-PTC per mol of enzyme. Although carbodiimides normally modify carboxyl groups in proteins, results from sulfhydryl group titrations and from reversible modification of sulfhydryl groups by methyl methanethiosulfonate suggest that two of the four cysteine residues of thymidylate synthase were modified by PTC.

Irreversible inhibition of thymidylate synthase by pyridoxine (B6) analogues.

Three vitamin B6 analogues have been synthesized and tested as inhibitors of thymidylate synthase. The compounds are: 4',5'-dichloro-, 4',5'-dibromo- and 4',5'-diiodo-pyridoxine. All three analogues inhibited the enzyme irreversibly. The kinetic data for the chloro- and bromo-analogues showed that a limiting rate of inhibition is approached as the inhibitor concentration is increased, which indicates that a reversible enzyme:inhibitor affinity complex is formed prior to the irreversible reaction. 4',5'-Dibromo-pyridoxine exhibited a greater binding affinity (lower Ki) for thymidylate synthase than 4',5'-dichloro-pyridoxine, and it also reacted faster to irreversibly inhibit the enzyme. The presence of the substrate dUMP (10 microM) completely protected thymidylate synthase from inhibition. These data suggest that the halogenated vitamin B6 analogues are active site-directed inhibitors of thymidylate synthase, which first bind reversibly to the catalytic site and then react irreversibly with the enzyme.

Inhibition of thymidylate synthase by glyceraldehyde 3-phosphate.

1. A number of common metabolites which had carbonyl and/or phosphate groups were tested for their ability to alter the activity of thymidylate synthase from Lactobacillus casei. Glyceraldehyde 3-phosphate was found to be an effective inhibitor of thymidylate synthase. 2. Glyceraldehyde 3-phosphate reversibly inhibited thymidylate synthase with a K1 of 12-13 microM; the inhibition was competitive with dUMP and noncompetitive with 5,10-methylenetetrahydrofolate which is consistent with an ordered addition of substrates.

Inhibition of thymidylate synthase by pyridoxal phosphate.

1. Pyridoxal phosphate (PLP) reversibly inhibited thymidylate synthase from Lactobacillus casei with a KI of 0.6-0.9 microM. 2. The inhibition was competitive with dUMP and noncompetitive with 5,10-methylenetetrahydrofolate which is consistent with an ordered addition of substrates. 3. The spectrum of PLP was altered by the addition of thymidylate synthase. The spectral changes suggest formation of a thiohemiacetal with an enzyme sulfhydryl group rather than Schiff base formation with a lysine side chain.

Purification and properties of alkaline phosphatase from boar seminal plasma.

An alkaline phosphatase was purified from boar seminal plasma using adsorption to calcium phosphate gel, gel filtration, and ion-exchange chromatography. The preparation gave a single band on SDS polyacrylamide electrophoresis. The enzyme was a non-specific alkaline phosphatase that hydrolysed pyrophosphate slowly and had no phosphodiesterase activity. The pH optimum was 10 and the Km was approximately 0.2 mM with p-nitrophenyl phosphate as substrate. The enzyme was a zinc metalloenzyme as indicated by the loss of activity when treated with o-phenanthroline and the restoration of activity by zinc and magnesium ions. It also lost activity when treated with thiols. Molecular weight estimates from SDS polyacrylamide gel electrophoresis and gel filtration suggest that the enzyme is a tetramer of identical subunits, each of which has a molecular weight of 68,000.

Microcomputer simulation of steady-state enzyme kinetics for educational purposes.

A BASIC program to assist the instruction of steady-state enzyme kinetics has been developed for the IBM PC microcomputer. Its purpose is to simulate laboratory experiments in order to minimize the time required to obtain kinetic data from which students deduce kinetic mechanisms and determine kinetic constants of enzyme-catalyzed reactions. The program randomly selects a kinetic scheme from various sequential, ping pong, and iso reaction sequences as well as values for the kinetic constants. The scheme and kinetic constants are unknown to the student at this time; the only thing he or she knows is the stoichiometry of the catalyzed reaction which can have two or three substrates and products. The student is prompted to enter values for concentrations of substrates and products; several different concentrations for each substrate and product can be entered in a single experiment. The program then calculates, displays and prints (if desired) the corresponding initial steady-state velocities. The student can perform as many experiments as desired until enough information is obtained to determine the kinetic mechanism and to calculate values for the kinetic constants.

Inactivation of thymidylate synthase by chlorine disinfectants.

The effects of two chlorine disinfectants, calcium hypochlorite (HTH) and 3-chloro-4,4-dimethyl-2-oxazolidinone (Agent I), on the activity of thymidylate synthase have been investigated. Although both disinfectants inactivated the enzyme, the following differences were observed: When the two disinfectants were used at the same total chlorine concentration, the rate and extent of inactivation were greater with Agent I than HTH. The substrate dUMP partially protected thymidylate synthase from inactivation by Agent I, but it did not appreciably protect against inactivation by HTH. Large changes in the ultraviolet spectrum of the enzyme occurred when it was treated with HTH, which suggests reactions with aromatic amino acid side chains; no spectral changes occurred when thymidylate synthase was treated with Agent I. Blocking the sulfhydryl groups of thymidylate synthase with sulfhydryl reagents prevented the irreversible inactivation of the enzyme by Agent I, but not by HTH.

Purification and properties of beta-N-acetyl-D-hexosaminidase from boar seminal plasma.

beta-N-Acetyl-D-hexosaminidase has been purified ca. 190-fold to homogeneity from boar seminal plasma. It catalyzed the hydrolysis of the p-nitrophenyl-N-acetyl derivatives of both beta-D-glucosaminide and beta-D-galactosaminide but was inactive with the o- or p-nitrophenyl glycosides of other monosaccharides. Its pH optimum was 4.5 and its KM was 1.5 mM with p-nitrophenyl-N-acetyl-beta-D-glucosamide as substrate. The enzyme was inhibited by mercuribenzoate compounds but not by iodoacetamide, 2,2'-dipyridyl disulfide, methylmethane thiosulfonate, nor N-ethylmaleimide. The active enzyme had mol. wt ca. 250,000 by Sephacryl S-300 chromatography. SDS electrophoresis showed single bands corresponding to subunit mol. wts ca. 62,000 and 107,000 depending on whether the enzyme had been denatured in the presence of 2-mercaptoethanol or not. These data suggest that the enzyme is a tetramer of identical subunits, pairs of which are held together by disulfide bonds.

The inactivation of thymidylate synthase by periodate.

Thymidylate synthase from methotrexate-resistant Lactobacillus casei rapidly lost about 90% of its catalytic activity when incubated with an equimolar concentration of IO4- at 0 degree C. Nearly complete inhibition resulted when the IO4- concentration was twice the enzyme concentration or higher. The inhibition reaction appeared to be pseudo-first-order with respect to enzyme when IO4- was in excess. The substrate dUMP, the product dTMP, and inorganic phosphate all protected the enzyme from inactivation by IO4-, with the order of effectiveness: dUMP greater than dTMP greater than phosphate. Deoxyuridine, which is not a substrate, did not protect the enzyme. Titrations with dithiobis(2-nitrobenzoate) (DTNB) showed that approximately 1.5 titratable SH groups were lost when thymidylate synthase was completely inhibited by IO4-. Essentially no reactivation occurred when periodate-inhibited enzyme was dialyzed against buffered 2-mercaptoethanol (ME) or dithiothreitol (DTT). Enzyme that had been treated with p-hydroxymercuribenzoate, DTNB, or methylmethanethiosulfonate prior to treatment with periodate could be completely reactivated with ME or DTT.

Oxidation of thymidylate synthase by inorganic compounds.

Thymidylate synthase from methotrexate-resistant Lactobacillus casei was rapidly and completely inactivated by low concentrations of permanganate, periodate, or potassium triiodide at 0 degree C. The enzyme was not inactivated to any appreciable extent by iodate, iodide, ferricyanate, iodosobenzoate, or hydrogen peroxide. The inactivation by permanganate was retarded by the substrate 2'-deoxyuridylate and, to a lesser extent, by phosphate. Titration of enzyme activity with permanganate showed that two moles of permanganate were required to completely inactivate one mole of thymidylate synthase.

Growth of Lactobacillus casei in deuterated media. Purification of deuterated thymidylate synthase and proton nuclear magnetic resonance studies.

The purification of thymidylate synthase from amethopterin-resistant Lactobacillus casei grown in a deuterated medium is described. The deuterated enzyme and the non-deuterated enzyme appear to be identical with respect to specific activity, pH optimum, electrophoretic mobility on polyacrylamide gels and ability to form ternary complexes with 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) and 5,10-methylenetetrahydrofolate. The deuterated enzyme remained stable at 25 degrees C for 8 h during the acquisition of 400 MHz 1H-NMR spectra and was stored at 5 degrees C for 3 months without loss of catalytic activity. The incorporation of deuterium was essentially complete as demonstrated by a comparison of the NMR spectra of deuterated and non-deuterated enzyme. Proton NMR data obtained with deuterated thymidylate synthase and 1H-FdUMP are in agreement with the fluorine-19 NMR studies of Lewis et al. (Lewis, C.A., Jr., Ellis, P.D. and Dunlap, R.B. (1980) Biochemistry 19, 116-123) which support the formation of a binary complex where the enzyme is covalently linked to carbon 6 of 5-fluoro-5,6-dihydro-2'-deoxyuridylate.

Inactivation of dihydrofolate reductase from Lactobacillus casei by diethyl pyrocarbonate.

The role of histidine residues of dihydrofolate reductase from Lactobacillus casei was investigated with diethyl pyrocarbonate. This enzyme has no cysteine residues and differs in this respect from many nicotinamide nucleotide dehydrogenases, which have catalytically important sulfhydryl groups. X-ray studies of this enzyme have shown that histidine residues are involved in substrate binding but not in proton transfer [Matthews et al. (1978) J. Biol. Chem. 253, 6946]. Dihydrofolate reductase was inactivated by diethyl pyrocarbonate; the second-order rate constant for the reaction was 29 M-1 min-1 at 0 degrees C. The difference spectrum of native and diethyl pyrocarbonate inactivated enzyme had a maximum near 242 nm, which indicated a reaction with histidine residues. The absence of any spectral difference near 280 nm indicated that diethyl pyrocarbonate had not reacted with tyrosine residues. Dihydrofolate reductase lost all of its enzymatic activity after about six of the seven histidine residues had been modified. No catalytic activity was lost during an initial rapid reaction with about four histidine residues, but a subsequent slower reaction involving an additional one or two residues was associated with the loss of activity. The enzyme was protected from inactivation by either of the substrates NADPH or dihydrofolate. In fact, treatment with diethyl pyrocarbonate in the presence of either substrate, but particularly with NADPH, resulted in substantially greater activity than that found with untreated enzyme. Treatment with 1 M hydroxylamine partially restored activity to dihydrofolate reductase that had been inactivated by diethyl pyrocarbonate.

The inactivation of thymidylate synthase by diethyl pyrocarbonate.

Thymidylate synthase (5,10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) from Lactobacillus casei was inactivated by treatment with diethyl pyrocarbonate. The inactivation was apparently due to the modification of a large proportion of the enzyme's histidine residues. Neither the substrate dUMP nor the product dTMP prevented inactivation by diethyl pyrocarbonate. The inactivated enzyme was not reactivated by treatment with hydroxylamine. These results indicate that histidine residues are involved in the maintenance of enzyme structure.

Kinetics of thymidylate synthase inhibition by disulfides.

The inactivation of thymidylate synthase (5,10-methylene-tetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) by a number of disulfides has been examined and found to be a second-order process. The apparent second-order rate constant was strongly influenced by the chemical structure of the disulfide. The data suggest that negatively charged functional groups decrease the reactivity of the disulfides and that positively charged groups enhance the reactivity. GSSG did not react with non-catalytic SH groups, since the number of SH groups of both GSSG-treated and untreated thymidylate synthase was the same. Several sulfhydryl compounds were tested for their ability to reactivate thymidylate synthase that had been inhibited by 2,2'-dithiodipyridine. Complete reactivation was obtained with either dithiothreitol or 2-mercaptoethanol. Reactivation by 2-mercaptoethanol was a second-order process.

The effects of platinum complexes on seven enzymes.

The effects of K2PtCl4, cis-Pt(NH3)2Cl2, and trans-Pt(NH3)2Cl2 on the activities of glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, dihydrofolate reductase, fructose-1,6-bisphosphate aldolase, catalase, tyrosinase, and peroxidase have been investigated. All of the enzymes which are thought to have essential sulfhydryl groups (glyceraldehyde-3-phosphate dehydrogenase, aldolase, and glucose-6-phosphate dehydrogenase) were significantly inhibited by K2PtCl4. The other four enzymes studied are not known to have essential sulfhydryl groups, and were not significantly affected by the Pt compounds under the conditions employed. Glyceraldehyde-3-phosphate dehydrogenase was the only enzyme inhibited by all three Pt compounds tested, with K2PtCl4 being the most effective and cis-Pt(NH3)2Cl2 the least effective inhibitor. Semilogarithmic plots of residual activity versus inhibition time indicated that the inhibition reactions were not simple first-order processes, except for the inhibition of glucose-6-phosphate dehydrogenase by K2PtCl4 which appeared to be first-order with respect to enzyme concentration.