Mevalonate-5-diphosphate decarboxylase: stereochemical course of ATP-dependent phosphorylation of mevalonate 5-diphosphate

Chicken liver mevalonate-5-diphosphate decarboxylase catalyzes the reaction of mevalonate 5-diphosphate (MVADP) with ATP to produce isopentenyl diphosphate, ADP, CO2, and inorganic phosphate. The overall reaction involves an anti elimination of the tertiary hydroxyl and carboxyl groups. To investigate the mechanism for transfer of the terminal phosphoryl group of ATP to the C-3 oxygen of MVADP, we have carried out the reaction using stereospecifically labeled (Sp)-adenosine 5'-O-(3-thio[3-17O2,18O]triphosphate) [( gamma-17O2,18O]ATP gamma S) in place of ATP. The configuration of the [17O,18O]thiophosphate produced was found to be Rp, corresponding to overall inversion of configuration at phosphorus in the thiophosphoryl group transfer step. This result is consistent with the direct transfer of the thiophosphoryl group from (Sp)-[gamma-17O2,18O]ATP gamma S to MVADP at the active site. Our result does not indicate the involvement of a covalent thiophosphoryl-enzyme on the reaction pathway.     

The stereochemical course of phosphoryl transfer catalysed by polynucleotide kinase (bacteriophage-T4-infected Escherichia coli B).

Polynucleotide kinase (bacteriophage-T4-infected Escherichia coli B) catalyses the transfer of the [gamma-16O,17O,18O]phosphoryl group from 5'[gamma(S)-16O,17O,18O]ATP to 3'-AMP with inversion of configuration at the phosphorus atom. The simplest interpretation of this observation is that the [gamma-16O,17O,18O]phosphoryl group is transferred directly from ATP to the co-substrate by an 'in-line' mechanism.     

The stereochemical course of phospho transfer catalyzed by adenylosuccinate synthetase. A reaction pathway via a phosphorylated intermediate with net inversion

The stereochemical course of phospho transfer in the reaction catalyzed by adenylosuccinate synthetase from rat muscle has been determined with chiral [gamma-17O,18O]GTP gamma S as a substrate. The stereochemical configuration of the product, inorganic thiophosphate, was determined by 31P NMR after the compound was stereospecifically incorporated into ATP beta S. The reaction goes with net inversion of configuration, which is the course for a single phospho transfer, even though 6-phospho-IMP is probably an intermediate on the normal reaction pathway (Liebermann, I. (1956) J. Biol. Chem. 223, 327-339). The breakdown of this intermediate goes by C-O bond cleavage and so is not a true phospho transfer step. Thus, inversion of configuration during the course of this ligase reaction is consistent with a single phospho transfer step in the overall reaction, the formation of the phosphorylated intermediate.     

Evidence that catalysis by yeast inorganic pyrophosphatase proceeds by direct phosphoryl transfer to water and not via a phosphoryl enzyme intermediate

In this work, we show that adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) is a substrate for yeast inorganic pyrophosphatase (PPase) (EC 3.6.1.1) and further, using chirally labeled [gamma-17O,18O]ATP gamma S, that enzyme-catalyzed hydrolysis to produce chiral inorganic thio[17O,18O]phosphate proceeds with inversion of configuration. Both the synthesis of chiral ATP gamma S and the determination of inorganic thiophosphate configuration were carried out as described by Webb [Webb, M. R. (1982) Methods Enzymol. 87, 301-316]. We also show in a single turnover experiment performed in H2(18)O that 1 mol each of 18O16O3P and 16O4P is produced per mol of inorganic pyrophosphate hydrolyzed, a strong indication that oxygen uptake to form inorganic phosphate on PPase catalysis of inorganic pyrophosphate hydrolysis comes directly from H2O. These two results provide strong evidence for the conclusion that PPase catalyzes inorganic pyrophosphate hydrolysis via a single-step direct phosphoryl transfer to water and does not involve formation of a phosphorylated enzyme intermediate.     

Stereochemical course of phosphokinases. The use of adenosine [gamma-(S)-16O,17O,18O]triphosphate and the mechanistic consequences for the reactions catalyzed by glycerol kinase, hexokinase, pyruvate kinase, and acetate kinase.

We report the synthesis of adenosine [gamma-(S)-16O,17O,18O]triphosphate, an isotopically labeled species of ATP that is chiral at the gamma-phosphoryl group, the configuration of which has been confirmed by independent stereochemical analysis. This molecule has been used as a substrate in the reactions catalyzed by glycerol kinase and by acetate kinase. The resulting samples of isotopically labeled sn-glycerol 3-phosphate and of acetyl phosphate have been used as substrates in the alkaline phosphatase mediated transfer of the chiral phosphoryl groups to (S)-propane-1,2-diol, whence the configuration at phosphorus has been determined [Abbott, S. J., Jones, S. R., Weinman, S. A., & Knowles, J. R. (1978) J. Am. Chem. Soc. 100, 2558]. It is shown that glycerol kinase and acetate kinase (and, by virtue of an earlier correlation, pyruvate kinase and hexokinase) proceed by pathways that result in inversion of the configuration at phosphorus. The sterochemical approach provides an access to the otherwise cryptic events that are involved in phosphoryl-group transfer within the ternary complexes of these kinases and their substrates.     

Stereochemical course of the reactions catalyzed by the bacterial phosphoenolpyruvate:glucose phosphotransferase system

The overall stereochemical course of the reactions leading to the phosphorylation of methyl alpha-D-glucopyranoside by the glucose-specific enzyme II (enzyme IIGlc) of the Escherichia coli phosphotransferase system has been investigated. With [(R)-16O,17O,18O]phosphoenolpyruvate as the phosphoryl donor and in the presence of enzyme I, HPr, and enzyme IIIGlc of the phosphotransferase system, membranes from E. coli containing enzyme IIGlc catalyzed the formation of methyl alpha-D-glucopyranoside 6-phosphate with overall inversion of the configuration at phosphorus (with respect to phosphoenolpyruvate). It has previously been shown that sequential covalent transfer of the phosphoryl group of phosphoenolpyruvate to enzyme I, to HPr, and to enzyme IIIGlc occurs before the final transfer from phospho-enzyme IIIGlc to the sugar, catalyzed by enzyme IIGlc. Because overall inversion of the configuration of the chiral phospho group of phosphoenolpyruvate implies an odd number of transfer steps, the phospho group has been transferred at least five times, and transfer from phospho-enzyme IIIGlc to the sugar must occur in two steps (or a multiple thereof). On the basis that no membrane protein other than enzyme IIGlc is directly involved in the final phospho transfer steps, our results imply that a covalent phospho-enzyme IIGlc is an intermediate during transport and phosphorylation of glucose by the E. coli phosphotransferase system.     

The stereochemical course of phosphoryl transfer catalyzed by herpes simplex virus type I-induced thymidine kinase

Herpes simplex virus type I (HSV-I)-induced thymidine kinase has been shown to catalyze phosphoryl transfer from adenosine 5'-[gamma-(S)-16O,17O,18O]triphosphate to thymidine with inversion of configuration at phosphorus. The simplest interpretation of this result is that phosphoryl transfer occurs by a single in-line group transfer between ATP and thymidine within the ternary enzyme complex.     

The stereochemical course of phosphoric residue transfer catalyzed by sarcoplasmic reticulum ATPase

The stereochemical course of the phosphoric residue transfer from ADP to water catalyzed by the (Mg2+ + Ca2+)-dependent ATPase of sarcoplasmic reticulum has been determined. For this determination, the preparation is described of ATP gamma S, stereospecifically labeled in the gamma-position with both 17O and 18O. After hydrolysis of this nucleotide, the analysis of the product inorganic [16O,17O,18O]thiophosphate showed that the reaction proceeded with retention of configuration at the gamma-phosphorus atom. This result is expected since a phosphoenzyme is well characterized for this ATPase and provides support for the hypothesis that each phosphate transfer step occurs with inversion. In this case, the formation and breakdown of the phosphoenzyme occur each with inversion leading to the retention observed for the whole reaction.     

Unambiguous stereochemical course of rabbit liver fructose bisphosphatase hydrolysis

The stereochemical course of rabbit liver fructose bisphosphatase (EC 3.1.3.11) was determined by hydrolyzing the substrate analogue (Sp)-[1-18O]fructose 1-phosphorothioate 6-phosphate in H(2)17O, incorporating the chiral, inorganic phosphorothioate product into adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), and analyzing the isotopic distribution of 18O in ATP beta S by 31P NMR. The result indicates that the 1-phosphoryl group is transferred with inversion of configuration. A series of single-turnover experiments ruled out an acyl phosphate intermediate in the hydrolysis. Consequently, fructose bisphosphatase catalyzes the hydrolysis of fructose 1,6-bisphosphate via a direct transfer of the phosphoryl moiety to water.     

Stereochemical course of thiophosphoryl transfer catalyzed by cytosolic phosphoenolpyruvate carboxykinase

Rat liver cytosolic phosphoenolpyruvate carboxykinase (PEPCK) utilizes inosine 5'-(3-thiotriphosphate) (ITP gamma S) as an excellent substrate, with Km and V values of 0.08 mM and 37 mumol min-1 (mg of protein)-1, respectively, compared with the corresponding values of 0.168 mM and 76 mumol min-1 (mg of protein)-1 for ITP. Thus, the V/Km values for the two substrates are the same. Reaction of (RP)-[gamma-18O2]ITP gamma S with oxalacetate catalyzed by cytosolic PEPCK produces (SP)-thio[18O]phosphoenolpyruvate. Therefore, thiophosphoryl transfer catalyzed by this enzyme proceeds with overall inversion of configuration at P. The reaction mechanism involves an uneven number of phosphotransfer steps, most likely a single step transfer between bound substrates. The results do not support the involvement of a phosphoryl enzyme intermediate in the mechanism.     

Investigations of the partial reactions catalyzed by pyruvate phosphate dikinase

The kinetic mechanism of pyruvate phosphate dikinase (PPDK) from Bacteroides symbiosus was investigated with several different kinetic diagnostics. Initial velocity patterns were intersecting for AMP/PPi and ATP/Pi substrate pairs and parallel for all other substrate pairs. PPDK was shown to catalyze [14C]pyruvate in equilibrium phosphoenolpyruvate (PEP) exchange in the absence of cosubstrates, [14C]AMP in equilibrium ATP exchange in the presence of Pi/PPi but not in their absence, and [32P]Pi in equilibrium PPi exchange in the presence of ATP/AMP but not in their absence. The enzyme was also shown, by using [alpha beta-18O, beta, beta-18O2]ATP and [beta gamma-18O, gamma, gamma, gamma-18O3]ATP and 31P NMR techniques, to catalyze exchange in ATP between the alpha beta-bridge oxygen and the alpha-P nonbridge oxygen and also between the beta gamma-bridge oxygen and the beta-P nonbridge oxygen. The exchanges were catalyzed by PPDK in the presence of Pi but not in its absence. These results were interpreted to support a bi(ATP,Pi) bi(AMP,PPi) uni(pyruvate) uni(PEP) mechanism. AMP and Pi binding order was examined by carrying out dead-end inhibition studies. The dead-end inhibitor adenosine 5'-monophosphorothioate (AMPS) was found to be competitive vs AMP, noncompetitive vs PPi, and uncompetitive vs PEP. The dead-end inhibitor imidodiphosphate (PNP) was found to be competitive vs PPi, uncompetitive vs AMP, and uncompetitive vs PEP. These results showed that AMP binds before PPi. The ATP and Pi binding order was studied by carrying out inhibition, positional isotope exchange, and alternate substrate studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of activation of phenylalanine and synthesis of P1, P4-bis(5'-adenosyl) tetraphosphate by yeast phenylalanyl-tRNA synthetase

The activation of L-phenylalanine by yeast phenylalanyl-tRNA synthetase using adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate is shown to proceed with inversion of configuration at P alpha of ATP. This observation taken together with the lack of positional isotope exchange when adenosine 5'-[beta,beta-18O2]triphosphate is incubated with the enzyme in the absence of phenylalanine and in the presence of the competitive inhibitor phenylalaninol indicates that activation of phenylalanine occurs by a direct "in-line" adenylyl-transfer reaction. In the presence of Zn2+, yeast phenylalanyl-tRNA synthetase also catalyzes the phenylalanine-dependent hydrolysis of ATP to AMP and the synthesis of P1,P4-bis(5'-adenosyl) tetraphosphate (Ap4A). With adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate, the formation of AMP and Ap4A is shown to occur with inversion and retention of configuration, respectively. It is concluded that phenylalanyl adenylate is an intermediate in both processes, Zn2+ promoting AMP formation by hydrolytic cleavage of the C-O bond and Ap4A formation by displacement at phosphorus of phenylalanine by ATP.     

A stereochemical study of the mechanism of activation of donor oligonucleotides by RNA ligase from bacteriophage T4 infected Escherichia coli

RNA ligase from bacteriophage T4 infected Escherichia coli catalyzes the activation of adenosine 3',5'-bisphosphate (representing the donor oligonucleotide) by adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate with retention of configuration at P alpha. Since single-step enzyme-catalyzed nucleotidyl transfer reactions proceed with inversion, this stereochemical result provides support for a double displacement mechanism involving an adenylyl-enzyme intermediate as proposed previously from isotope exchange experiments.     

The stereochemical course of yeast hexokinase-catalysed phosphoryl transfer by using adenosine 5''[gamma(S)-16O,17O,18O]triphosphate as substrate.

Adenosine 5'[gamma(S)-16O, 17O, 18O]triphosphate has been synthesized and used to determine the stereochemical course of phosphoryl transfer catalysed by yeast hexokinase. The chirality at phosphorus of the D-glucose 6-[16O,17O,18O]phosphate formed was analysed, after cyclization and methylation, by 31P n.m.r. spectroscopy. The phosphoryl transfer was found to occur with inversion of configuration, with a stereoselectivity in excess of 94%. The simplest interpretation of this result is that the phosphoryl group is transferred between substrates in the enzyme-substrate ternary complex by an 'in line' mechanism.     

Stereochemical probes of the argininosuccinate synthetase reaction

The stereochemical course of the argininosuccinate synthetase reaction has been determined. The SP isomer of [alpha-17O,alpha-18O,alpha beta-18O]ATP is cleaved to (SP)-[16O,17O,18O]AMP by the action of argininosuccinate synthetase in the presence of citrulline and aspartate. The overall stereochemical transformation is therefore net inversion, and thus the enzyme does not catalyze the formation of an adenylylated enzyme intermediate prior to the synthesis of citrulline adenylate. The RP isomer of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) is a substrate in the presence of Mg2+, but the SP isomer is a substrate when Cd2+ is used as the activating divalent cation. Therefore, the lambda screw sense configuration of the beta,gamma-bidentate metal--ATP complex is preferred by the enzyme as the actual substrate. No significant discrimination could be detected between the RP and SP isomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) when Mg2+ or Mn2+ are used as the divalent cation. Argininosuccinate synthetase has been shown to require a free divalent cation for full activity in addition to the metal ion needed to complex the ATP used in the reaction.     

The stereochemical course of phosphoric residue transfer catalyzed by beef heart mitochondrial ATPase

The stereochemical course of phosphoric residue transfer has been determined for beef heart mitochondrial ATPase. When aden 5'-(3-thiotriphosphate), stereospecifically labeled with 18O in the gamma position, was hydrolyzed in [17O]water in the presence of the ATPase, the product inorganic [16O, 17O, 18O]thiophosphate was chiral. The configuration of the product showed that the hydrolysis had proceeded with inversion at the gamma-phosphorus atom. This result suggests that there is a direct, in-line transfer of the phosphoric residue between ADP and water and that there is no phosphoenzyme intermediate.     

The stereochemical course of D-glyceraldehyde-induced ATPase activity of glycerokinase from Escherichia coli

D-Glyceraldehyde-induced hydrolysis of adenosine (R)-5'-[gamma-17O,18O,thio]triphosphate catalysed by glycerokinase from Escherichia coli gives inorganic [16O,17O,18O]thiophosphate with the (S)-configuration, showing that the reaction proceeds with inversion of configuration at phosphorus. This result provides powerful support for the chemically most plausible mechanism, namely, that the hydrate of D-glyceraldehyde is the effective substrate which after phosphorylation or thiophosphorylation eliminates inorganic phosphate or inorganic thiophosphate, respectively, with regeneration of D-glyceraldehyde.     

Kinetic mechanism and metabolic role of pyruvate phosphate dikinase from Entamoeba histolytica

The kinetic mechanism and the metabolic role of pyruvate phosphate dikinase from Entamoeba histolytica were investigated. The initial velocity patterns in double reciprocal plots were parallel for the phosphoenolpyruvate/AMP and phosphoenolpyruvate/pyrophosphate substrate pairs and intersecting for the AMP/pyrophosphate pair. This suggests a kinetic mechanism with two independent reactions. The rate of ATP synthesis at saturating and equimolar concentrations of phosphoenolpyruvate, AMP, and pyrophosphate was inhibited by phosphate, which is consistent with an ordered steady-state mechanism. Enzyme phosphorylation by [(32)P(i)]pyrophosphate depends on the formation of a ternary complex between AMP, pyrophosphate, and pyruvate phosphate dikinase. In consequence, the reaction that involves the AMP/pyrophosphate pair follows a sequential steady-state mechanism. The product inhibition patterns of ATP and phosphate versus phosphoenolpyruvate were noncompetitive and uncompetitive, respectively, suggesting that these products were released in an ordered process (phosphate before ATP). The ordered release of phosphate and ATP and the noncompetitive inhibition patterns of pyruvate versus AMP and versus pyrophosphate also supported the sequential kinetic mechanism between AMP and pyrophosphate. Taken together, our data provide evidence for a uni uni bi bi pingpong mechanism for recombinant pyruvate phosphate dikinase from E. histolytica. The Delta G value for the reaction catalyzed by pyruvate phosphate dikinase (+2.7 kcal/mol) determined under near physiological conditions indicates that the synthesis of ATP is not thermodynamically favorable in trophozoites of E. histolytica.     

The stereochemical course of thiophosphoryl group transfer catalyzed by T4 polynucleotide kinase

The stereochemical course of the phosphoryl transfer reaction catalyzed by T4 polynucleotide kinase has been determined using the chiral ATP analog, (Sp)-adenosine-5'-(3-thio-3-[18O]triphosphate). T4 polynucleotide kinase catalyzes the transfer of the gamma-thiophosphoryl group of (Sp)-adenosine-5'-(3-thio-3-[18O]triphosphate) to the 5'-hydroxyl group of ApA to give the thiophosphorylated dinucleotide adenyl-5'-[18O]phosphorothioate-(3'-5')adenosine. A sample of adenyl-5'-[18O]phosphorothioate-(3'-5')adenosine was subjected to venom phosphodiesterase digestion. The resulting adenosine-5'-[18O]phosphorothioate was shown to have the Rp configuration, thus indicating that the thiophosphoryl transfer reaction occurs with overall inversion of configuration of phosphorus.