Enzyme kinetics in mammalian cells. 3. Regulation of activities of galactokinase, galactose-1-phosphate uridyl transferase and uridine diphosphogalactose-4-epimerase in human erythrocytes

The sequential enzyme assay as previously described has been used to study various effects on the three enzymes in human red cells involved in the phosphorylation of galactose: galactokinase, galactose-1-phosphate uridyl transferase and uridine diphospho-galactose-4-epimerase.

• 1 Enzyme activities in undiluted lysates appear to reflect the respective activities in whole cells.
• 2 Added extracellular Gal-1-P, G-1-P, UDPGal and UPDG do not affect enzyme activities in whole cells.
• 3 The kinase and transferase enzymes do not appear to be associated with the membrane fraction of the red cells.
• 4 Galactokinase activity is inhibited by G-6-P and Gal-1-P, but not by glucose, G-1-P, UDPG, UDPGal, UTP or NAD⁺. It is inhibited by ATP and ADP in high concentration.
• 5 Galactose-1-phosphate uridyl transferase activity is inhibited by G-1-P, G-6-P, UDPG, UDPGal, ATP, and ADP. It is not affected by UTP, NAD⁺, or galactose.
• 6 Uridine diphospho-galactose-4-epimerase activity is inhibited by UDPG, ATP, ADP, UTP and NADH. It is stimulated by NAD⁺ and possibly by Gal-1-P. It is unaffected by G-1-P, G-6-P.
• 7 The rates of the three reactions decrease with decreasing temperature. The activities of transferase and epimerase are inactivated at the same rate, the kinase activity is inactivated more slowly.
• 8 Dilution experiments indicate the presence in lysates of a pool of UDPG (or, possibly UDPGal) which regulates the activities transferase and the epimerase enzymes.
• 9 Results of dilution experiments suggest that the radioactive product of the transferase enzyme is different from commercially available UDPGal-u-¹⁴C.
• 10 ATP, UTP and UDPG interact with some substance(s) in the red cell lysate to cause a time dependent inactivation of the epimerase. These interactions are the result of glucose metabolism.

     

Purification and steady state kinetic mechanism of glycogen synthase-D from human polymorpho-nuclear leukocytes

Summary The authors' work on the purification and steady state kinetic investigation of the enzyme glycogen synthase D (UDP-glucose: glycogen 4--glucosyl-transferase, EC 2.4.1.11) from human polymorphonuclear leukocytes is reviewed. The main features of the kinetic mechanism for catalysis of the reaction UDPG + glycogen_n UDP + glycogen_(n+1) are: (i) Lineweaver-Burk plots in both substrates are linear, exhibiting intersecting patterns; (ii) UDP is a competitive, respectively noncompetitive, inhibitor towards the substrates UDPG and glycogen; (iii) the essential activator glucose-6-phosphate (G-6-P) showed an intersecting pattern towards glycogen and an equilibrium ordered pattern towards UDPG. These features identify in this case the mechanism as a rapid equilibrium random bi-bi mechanism, with G-6-P adding to the enzyme prior to the substrate UDPG. New results on the influence of the modifiers NaCl, Ca⁺⁺, Mn⁺⁺, Mg⁺⁺, HPO₄ ^–-, SO₄ ^–-, and ATP on the enzyme are reported. Interpreting the observations in terms of the established mechanism, the following results are obtained: The effect of salt (NaCl) is nonspecific and fairly small, probably reflecting a general action of the electrolyte medium on the conformation of the enzyme. Divalent cations affect only the rate limiting step, i.e. the interconversion of the quaternary enzyme-substrate-activator complexes. The anions interact exclusively with the G-6-P binding site of the enzyme. The dissociation constants for the enzyme-modifier complexes are determined, and a kinetic mechanism for the action of the anions is proposed, leading to activation or inhibition, depending on the concentration of G-6-P.An invited article     

Involvement of sucrose synthase in sucrose catabolism

Maize scutellum slices incubated in water utilized sucrose at a maximum rate of 0.12,μmol/min per g fr. wt of slices. When slices were incubated in DNP, there was a three-fold increase in the rate of sucrose utilization. Sucrose breakdown in higher plants can be achieved by pathways starting with either invertase or sucrose synthase (SS). Invertase activity in scutellum homogenates was found only in the cell wall fraction, indicating that SS was responsible for sucrose breakdown in vivo. SS in crude scutellum extracts broke down sucrose to fructose and UDPG at 0.39,μmol/min per g fresh wt of slices. The UDPG formed was not converted to UDP + glucose, UMP + glucose-1-P, UDP + glucose-1-P or broken down by any other means by the crude extract in the absence of PPi. In the presence of PPi, UDPG was broken down by UDPG pyrophosphorylase which had a maximum activity of 26 μmol/min per g fr. wt of slices. Levels of PPi in the scutellum could not be measured using the UDPG pyrophosphorylase: phosphoglucomutase: glucose-6-P dehydrogenase assay because they were too low relative to glucose-6-P which interferes in the assay. An active inorganic pyrophosphatase was present in the scutellum extract which could prevent the accumulation of PPi in the cytoplasm. ATP pyrophosphohydrolase, which hydrolyses ATP to AMP and PPi, was found in the soluble portion of the scutellum extract. The enzyme activity was increased by fructose-2,6-bisP and Ca²⁺. In the presence of both activators, enzyme activity was 1.1 μmol/min per g fr. wt of slices, a rate sufficient to supply PPi for the breakdown of UDPG. These results indicate that sucrose breakdown in maize scutellum cells occurs via the SS: UDPG pyrophosphorylase pathway.     

PRODUCTION AND UTILIZATION OF GLUCOSE 1-PHOSPHATE IN THE EGGS OF THE SEA URCHIN ANTHOCIDARIS CRASSISPINA

Concentrations of G1P, G6P, UDPG, UTP and PPi were measured in the eggs of the sea urchin, Anthocidaris crassispina. Activities of phosphorylase a (EC 2.4.1.1), phosphoglucomutase (EC 2.7.5.1), UDPG pyrophosphorylase (EC 2.7.7.9) and pyrophosphatase (EC 3.6.1.1) were also estimated. Levels of G1P and G6P increase following fertilization, but concentrations of UDPG and UTP in unfertilized eggs are very similar to those in fertilized eggs. PPi is undetectable. In unfertilized and fertilized eggs, the G1P level is very low as compared with the G6P level and is far less than that expected from the equilibrium constant in a reaction catalyzed by phosphoglucomutase. Since the phosphoglucomutase activity is higher by about 20 times than the phosphorylase a activity, G1P is probably produced in the reverse reaction, catalyzed by phosphoglucomutase, rather than in the reaction catalyzed by phosphorylase. The G1P thus produced seems to be utilized thoroughly in the reaction catalyzed by UDPG pyrophosphorylase. The reaction seems to be irreversible and tends to go to UDPG production in sea urchin eggs, since the PPi level is negligible due to high pyrophosphatase activity. The utilization of G1P in the reaction catalyzed by UDPG pyrophosphorylase seems to keep the G1P level low.     

PPi-dependent phosphofructotransferase (phosphofructokinase) activity in the mollicutes (mycoplasma) Acholeplasma laidlawii.

A PPi-dependent phosphofructotransferase (PPi-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.90) which catalyzes the conversion of fructose 6 phosphate (F-6-P) to fructose 1,6-bisphosphate (F-1, 6-P2) was isolated from a cytoplasmic fraction of Acholeplasma laidlawii B-PG9 and partially purified (430-fold). PPi was required as the phosphate donor. ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, dUTP, ITP, TTP, ADP, or Pi could not substitute for PPi. The PPi-dependent reaction (2.0 mM PPi) was not altered in the presence of any of these nucleotides (2.0 mM) or in the presence of smaller (less than or equal to 300 microM) amounts of fructose 2,6-bisphosphate, (NH4)2SO4, AMP, citrate, GDP, or phosphoenolpyruvate. Mg2+ and a pH of 7.4 were required for maximum activity. The partially purified enzyme in sucrose density gradient experiments had an approximate molecular weight of 74,000 and a sedimentation coefficient of 6.7. A second form of the enzyme (molecular weight, 37,000) was detected, although in relatively smaller amounts, by using Blue Sepharose matrix when performing electrophoresis experiments. The back reaction, F-1, 6-P2 to F-6-P, required Pi; arsenate could substitute for Pi, but not PPi or any other nucleotide tested. The computer-derived kinetic constants (+/- standard deviation) for the reaction in the PPi-driven direction of F-1, 6-P2 were as follows: v, 38.9 +/- 0.48 mM min-1; Ka(PPi), 0.11 +/- 0.04 mM; Kb(F-6-P), 0.65 +/- 0.15 mM; and Kia(PPi), 0.39 +/- 0.11 mM. A. laidlawii B-PG9 required PPi not only for the PPi-phosphofructotransferase reaction which we describe but also for purine nucleoside kinase activity. a dependency unknown in any other organism. In A. laidlawii B-PG9, the PPi requirement may be met by reactions in this organism already known to synthesize PPi (e.g., dUTPase and purine nucleobase phosphoribosyltransferases). In almost all other cells, the conversion of F-6-P to F-1,6-P2 is ATP dependent, and the reaction is generally considered to be the rate-limiting step of glycolysis. The ability of A. laidlawii B-PG9 and one other acholeplasma to use PPi instead of ATP as an energy source may offer these cytochrome-deficient organisms some metabolic advantage and may represent a conserved metabolic remnant of an earlier evolutionary process.     

The Sucrose-synthesizing Enzyme in the Beans

The properties of UDPG → fructose transglucosylase, partially purified from immature soybeans or broad beans, were investigated. Magnesium ions had a stimulating effect on this enzyme. Evidence was presented to show that UDPG, the glucosyl group donor for sucrose synthesis, was regenerated from UDP and G-1-P in the presence of ATP.     

Uridine diphosphate glucose synthase from calf liver: determinants of enzyme activity in vitro.

The reaction catalyzed by calf liver uridine diphosphate glucose synthase (pyrophosphorylase) (EC 2.7.7.9; UTP + glucose 1-phosphate = UDP-glucose + PPi) is an example of an enzymic reaction in which a nucleoside triphosphate other than ATP is the immediate source of metabolic energy. Kinetic properties of the enzyme, acting in the direction of UCP-glucose formation were investigated in vitro. The reaction was inhibited by UDP-glucose (0.072), Pi (11), UDP (1.6), UDP-xylose (0.87), UDP-glucuronate (1.3), and UDP-galacturonate (0.95). The numbers in parentheses indicate the concentration (mM) required for half-maximal inhibition under the conditions used. Other compounds tested, including ATP, ADP, and AMP, had no effect. Over a range of concentrations of UTP (0.04-0.8 MM) and UDP-glucose (0.05-0.03 mM), the reaction rate was more dependent on the concentration ratio [UDP-glucose]/[UTP] than on the absolute concentration of either compound. Comparison of the kinetic properties in vitro with estimates of metabolite levels in vivo suggests that (1) the enzyme operates in a range far from its maximal rate, and (2) the concentrations of glucose 1-phosphate and Pi and the ratio [UDP-glucose]/[UTP] may be the most important determinants of UDP-glucose synthase activity.     

水稻叶片蔗糖磷酸合成酶的一些特性

水稻叶片粗提液经硫酸铰分部沉淀、DE 52纤维素及 Sephadex G—200柱层析,得到较纯的蔗糖磷酸合成酶。该酶的最适 PH约7.0;UTP,UDP,ATP能明显地抑制其酶活;UTP是该酶UDPG的竞争性抑制剂,Mg~( )对它有促进作用;G6P则无影响。酶的两个底物F6P及UDPG的饱和动力学曲线分别为双曲线型和S型;K_m(F6P)=0.93 mmol/L;K_m(UDPG)=20.0 mmol/L;V_m(F6P)=83.3 nmol Suc mg~(-1)Protein min~(-1);V_m(UDPG)=333 nmol Suc mg~(-1)protein min~(-1);Hill(F6P)=1.0,Hill(UDPG)=1.4。水稻叶片蔗糖磷酸合成酶的活性受 ATP,UTP,UDP,UDPG等因素的调节。水稻叶片中蔗糖合成酶的总活力大于或等于蔗糖磷酸合成酶。     

Effects of Pyrophosphate on Formation of Nucleotide Derivatives

It was found that CDP-choline was formed with good yield from 5′-CMP and choline phosphate or choline chloride by yeast cells. The effects of pyrophosphate (PP_i) on the formation of UDPG, GDPM and CDP-choline from respective nucleoside monophosphate by yeast cells were studied. By the addition of PP_i to the reaction mixture, the phosphorylation of G-6-P from glucose was inhibited and then the phosphorylation of nucleoside monophosphates was restrained. Such inhibition was reversed by the decomposition of PP_i by inorganic pyrophosphatase of yeast cells. The addition of PP_i after the formation of nucleotide derivatives caused the accumulation of UTP and GTP and molar yields from nucleotide as substrate was about 80%. But that of CTP was a little in the reaction system of CDP-choline synthesis. Further, this method seems to be suitable for the accumulation of sugar-1-phosphates.     

Nucleoside triphosphate pyrophosphatase of rabbit matrix vesicles, a mechanism for the generation of inorganic pyrophosphate in epiphyseal cartilage

Inorganic pyrophosphate (PPi) may be important in the regulation of mineralisation but its origin in epiphyseal cartilage is ill-defined. Nucleoside triphosphate pyrophosphatase is one potential source, as this enzyme catalyses the formation of PPi from nucleoside triphosphates. This enzyme has been identified in matrix vesicles derived from rabbit epiphyseal cartilage and a method developed to measure the activity using ATP as substrate in intact matrix vesicles under relatively physiological conditions. The enzyme had a high affinity for ATP (Km less than 10 microM) and was also active towards GTP, CTP and UTP. Disruption of the matrix vesicle membrane by sonication failed to alter the activity. Treatment of sonicated matrix vesicles with Triton X-100 increased the activity which may indicate a direct effect of the detergent on the enzyme. Activity towards ATP was inhibited substantially by ADP and AMP and by another potential substrate beta,gamma-methyleneadenosine 5'-triphosphate. Dichloromethylene bisphosphonate, an analogue of the product PPi, inhibited the activity to a lesser extent. Two other potential substrates, NADP+ and thymidine 5'-monophosphate p-nitrophenyl ester were only weakly inhibitory as was 1-hydroxyethylidene 1,1-bisphosphonate. These results imply that nucleoside triphosphates are the substrates in vivo and the inhibitory effects of ADP and AMP suggest mechanisms whereby this activity could be regulated.     

Kinetics of the D and I glycogen synthetase forms and their interconversion, in the sea scallop Pecten maximus.

In extracts from the adductor muscle of the shell-fish, Pecten maximus, glycogen synthetase (EC.2.4.1.11) was found. The enzyme occurs predominantly as D form (glucose-6-P dependent for activity). An I form (G-6-P independent) was also present. Kinetics of glycogen synthetase showed that the Ka for G-6-P in the D form was 10 fold higher than in the I form. Both forms of glycogen synthetase were interconverted through reactions catalyzed by phosphatase and kinase enzymes respectively. Glucose-6-P and Mg+2 must be present to stabilize glycogen synthetase and to activate the synthetase D phosphatase, found in the 90,000 X g protein-glycogen complex. The conversion of synthetase D to I was inhibited by F-, glycogen, ATP and UTP. When F- was present the effect of G-6-P on synthetase and phosphatase suggested that conversion involved the existence of more than a single glycogen synthetase phosphatase enzyme. ATP and Mg+2 were necessary for the conversion of synthetase I to D, and the conversion was stimulated by cAMP.     

Purification and kinetic properties of human erythrocyte Mg2+-dependent inorganic pyrophosphatase

Inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) from human erythrocyte hemolysates has been purified up to 10 000-fold. The purified enzyme is homogenous and has a specific activity of 79.75 mumol PPi hydrolysed.min-1.mg-1 at pH 8 and 37 degrees C. It was confirmed that it is a dimer with a molecular weight of 42 000, composed of two identical protomers. From kinetic studies, it is proposed that human erythrocyte inorganic pyrophosphatase activity depends on free Mg2+ concentration in different ways. This ion constitutes part of the substrate (the Mg.PPi complex; Km = 1.4.10(-4) M) and probably acts as an allosteric activator (kinetic activation constant: KMg2+a = 7.5.10(-4) M). Equilibrium binding studies performed in the absence of PPi showed 4 binding sites for Mg2+, all having the same high affinity (dissociation constant: KMg2+d = 4.10(-6) M). Since the concentration of free Mg2+ in red blood cells is very low and may vary with the oxygenation state, it is likely that in vivo erythrocyte pyrophosphatase activity is regulated.     

Evidence for a novel metabolic pathway of (ADP-ribose)n: pyrophosphorolysis of ADP-ribose in HeLa S3 cell nuclei

ADP-ribose liberated from (ADP-ribose)n by the action of (ADP-ribose)n glycohydrolase was converted to ATP and ribose 5-phosphate (ribose 5-P) in the presence of pyrophosphate (PPi) in HeLa S3 cell nuclei. This reaction was reversible and dependent on the simultaneous presence of ADP-ribose, PPi, Mg2+, and nuclei. These results suggest the presence of a novel enzyme in the nuclei, designated as ADP-ribose pyrophosphorylase, which catalyzes the reaction shown in Equation 1. ADP-ribose + PPi in equilibrium ATP + Ribose 5-P (1) This reaction could represent a pathway for the biosynthesis of ATP from (ADP-ribose)n in eukaryotic cell nuclei.     

Pyrophosphorylases in Solanum tuberosum (IV. Purification,Tissue Localization,and Physicochemical Properties of UDP-Glucose Pyrophosphorylase)

The enzyme UDP-glucose pyrophosphorylase (UGPase) from potato (Solanum tuberosum L. cv Norchip) tubers was purified 177-fold to near homogeneity and to a specific activity of 1099 international units/mg of protein. The molecular mass of the purified enzyme was 53 kD as determined by SDS-PAGE and gel filtration. Immunological and activity assays detected UGPase at similar levels in potato stems, stolons, and tubers. Leaves and roots contained lower levels of UGPase activity and protein. Lineweaver-Burk plots for substrates inorganic pyrophosphate and UDP-glucose were linear in the pyrophosphorolytic direction, yielding Km values of 0.13 and 0.14 mM, respectively. However, Lineweaver-Burk plots for the substrates glucose-1-P and UTP were biphasic in nature when UGPase was assayed in the direction of UDP-glucose synthesis. At physiological substrate concentrations (i.e. from 0.05-0.20 mM), Km values of 0.08 mM (glucose-1-P) and 0.12mM (UTP) were obtained. When substrate concentrations increased above 0.20 mM, Km values increased to 0.68 mM (glucose-1-P) and 0.53 mM (UTP). These kinetic patterns of potato UGPase suggest a "negative cooperative effect" (A. Conway, D.E. Koshland, Jr. [1968] Biochemistry 7: 4011-4022) with respect to the substrates glucose-1-P and UTP. The biphasic substrate saturation curves were similar to the kinetics of the dimeric form of UGPase purified from Salmonella typhimurium (T. Nakae [1971] J Biol Chem 246: 4404-4411). The in vivo significance of the enzyme's "negative cooperativity" in the direction of UDP-glucose synthesis and potato sweetening is discussed.     

Enzymatic synthesis of UDP-GlcN by a two step hollow fiber enzyme reactor system

UDP-GlcN was synthesized from GlcN and UTP by a two step hollow fiber enzyme reactor method. In step 1, GlcN was converted to GlcN 6-P and then to GlcN 1-P by hexokinase and phosphoglucomutase, respectively, and UTP was used as the phosphate donor. In step 2, GlcN 1-P was converted to UDP-GlcN by UDP glucose pyrophosphorylase. All the enzymes required for the synthesis of UDP-GlcN were enclosed in hollow fiber bundles which allow for the free diffusion of substrates and products across the membranes to and from the enzymes, allow for the reutilization of the enzymes, and simplify the isolation of the product, UDP-GlcN. We show that both UTP and GlcN 6-P are inhibitors of the yeast UDPG pyrophosphorylase and therefore their concentrations must be regulated to obtain maximum yields of UDP-GlcN. The UDP-GlcN produced can be N-acetylated with [14C]acetic anhydride to produce UDP-[14C]GlcNAc. This method can also be used to synthesize [32P]UDP-GlcN and [32P]UDP-GlcNAc from [alpha-32P]UTP and GlcN 1-P.     

Evidence for the participation of independent translocation for phosphate and glucose 6-phosphate in the microsomal glucose-6-phosphatase system. Interactions of the system with orthophosphate, inorganic pyrophosphate, and carbamyl phosphate

The interactions of Pi, PPi, and carbamyl-P with the hepatic glucose-6-phosphatase system were studied in intact and detergent-disrupted microsomes. Penetration of PPi and carbamyl-P into intact microsomes was evidenced by their reactions with the enzyme located exclusively on the luminal surface. Lack of effects of carbonyl cyanide m-chlorophenylhydrazone and valinomycin + KCl indicated that pH gradients and/or membrane potentials that could influence the kinetics of the system are not generated during metabolism of PPi and glucose-6-P by intact microsomes. With disrupted microsomes, only competitive interactions were seen among glucose-6-P, Pi, PPi, and carbamyl-P. With intact microsomes, Pi, PPi, and carbamyl-P were relatively weak, noncompetitive inhibitors of glucose-6-phosphatase, and PPi hydrolysis was inhibited competitively by Pi and carbamyl-P but noncompetitively by glucose-6-P. Analysis of the kinetic data in combination with findings from other studies that a variety of inhibitors of the glucose-6-P translocase (T1) does not affect PPi hydrolysis provide compelling evidence that permeability of microsomes to Pi, PPi, and carbamyl-P is mediated by a second translocase (T2). Some properties of the microsomal anion transporters are described. If the characteristics of the glucose-6-phosphatase system as presently defined in intact microsomes apply in vivo, glucose-6-P hydrolysis appears to be the predominant, if not the exclusive, physiologic function of the system. Both the "noncompetitive character" and the relative ineffectiveness of Pi as an inhibitor of glucose-6-phosphatase of intact microsomes result from the rate limitation imposed by T1 that prevents equilibration of glucose-6-P across the membrane. In microsomes from fed rats, where T1 is less rate restricting, about one-half as much Pi was required to give 50% inhibition compared with microsomes from fasted or diabetic rats. Thus, any treatment or agent that alters the kinetic relationship between transport and hydrolysis of glucose-6-P (e.g. endocrine or nutritional status) is an essential consideration in analyses of kinetic data for the glucose-6-phosphatase system.     

Observation of uridine triphosphate:glucose-1-phosphate uridylyltransferase activity in maize root tips by saturation transfer 31P-NMR. Estimation of cytoplasmic PP

Saturation transfer 31P nuclear magnetic resonance was used to estimate the unidirectional rate of phosphorus exchange between Glc-1-P and UDPGlc in maize root tips. The rate was determined to be approx. 4 mumol.min-1 per g fresh weight. This estimated rate is much higher than net rates of other reactions in glucose metabolism (e.g., more than 10-times faster than the maximal glycolytic flux in this tissue). Furthermore, exchange between Glc-1-P and UDPGlc was not significantly inhibited by the metabolic poison KCN. We conclude that the unidirectional rate of conversion of Glc-1P to UDPGlc is much faster than the net rate of UDPGlc synthesis--the UTP:Glc-1-P uridylyltransferase reaction is near-equilibrium in vivo. From the equilibrium constant for this transferase reaction and the concentrations of Glc-1-P, UTP and UDPGlc, the level of cytoplasmic PPi was estimated to be approx. 10 nmol.g-1.     

Pyrophosphate may be involved in regulation of bacterial glycogen synthesis

Inorganic pyrophosphate is a potent inhibitor of the enzyme that catalyzes synthesis of the glucosyl donor for Escherichia coli glycogen synthesis, ADP-glucose pyrophosphorylase. The Ki is determined to be 40 microM and the substrate ATP, the activator, fructose 1,6-P2 or the allosteric inhibitor, AMP do not greatly affect the inhibition. PPi exhibits mixed type inhibition with the other substrate, glucose 1-P. The potential regulation of glycogen synthesis by PPi is discussed.     

An Enzymatic Preparation of UDP (U-13C) Glucose

Uniformly labeled uridine diphosphoglucose (UDP(U-¹³C)G) was prepared by a two-step enzymatic synthesis. (U-¹³C) G-6-P was prepared quantitatively by incubating (U-¹³C) glucose, ATP, MgS0₄, and hexokinase. UDP(U-¹³C) Glucose was prepared by incubation of (U-¹³C)G-6-P with UDPG pyrophosphorylase, phosphoglucomutase, inorganic pyrophosphatase, UTP, and glucose-1, 6-diphosphate in pH 7.5, 100 mM Tris-HCl buffer. After purification over Biogel P-2 and subsequent preparative HPLC, UDP (U-¹³C)G was obtained in 50% yield. UDP(U-¹³C)G was characterized by ¹³C NMR and FAB-MS.     

Inhibition of glyceraldehyde-3-phosphate dehydrogenase by pentalenolactone: kinetic and mechanistic studies

Incubation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with the antibiotic pentalenolactone (1) resulted in time-dependent, irreversible inhibition of GAPDH. The kinetics of inactivation were biphasic, exhibiting an initial rapid phase and a slower second phase. Pentalenolactone methyl ester (2) also irreversibly inactivated GADPH, albeit at a slower rate and with a higher KI. The substrate glyceraldehyde-3-phosphate (G-3-P) afforded protection against inactivation by 1, whereas the presence of NAD+ in the incubation mixture stimulated the inactivation by increasing the apparent affinity of the enzyme for the inhibitor. In steady-state kinetic experiments, 1 acted as a competitive inhibitor of GAPDH with respect to G-3-P but exhibited uncompetitive inhibition with respect to NAD+. Inactivation of NAD+-free apo-GAPDH by 1 showed simple pseudo-first-order kinetics. By titrating the free thiol residues of partially inactivated GAPDH, it was found that both pentalenolactone and pentalenolactone methyl ester react with all four Cys-SH residues of the tetrameric GAPDH.