Triosephosphate isomerase: removal of a putatively electrophilic histidine residue results in a subtle change in catalytic mechanism

An important active-site residue in the glycolytic enzyme triosephosphate isomerase is His-95, which appears to act as an electrophilic component in catalyzing the enolization of the substrates. With the techniques of site-directed mutagenesis, His-95 has been replaced by Gln in the isomerase from Saccharomyces cerevisiae. The mutant isomerase has been expressed in Escherichia coli strain DF502 and purified to homogeneity. The specific catalytic activity of the mutant enzyme is less than that of wild type by a factor of nearly 400. The mutant enzyme can be resolved from the wild-type isomerase on nondenaturing isoelectric focusing gels, and an isomerase activity stain shows that the observed catalytic activity indeed derives from the mutant protein. The inhibition constants for arsenate and for glycerol phosphate with the mutant enzyme are similar to those with the wild-type isomerase, but the substrate analogues 2-phosphoglycolate and phosphoglycolohydroxamate bind 8- and 35-fold, respectively, more weakly to the mutant isomerase. The mutant enzyme shows the same stereospecificity of proton transfer as the wild type. Tritium exchange experiments similar to those used to define the free energy profile for the wild-type yeast isomerase, together with a new method of analysis involving 14C and 3H doubly labeled substrates, have been used to investigate the energetics of the mutant enzyme catalyzed reaction. When the enzymatic reaction is conducted in tritiated solvent, the mutant isomerase does not catalyze any appreciable exchange between protons of the remaining substrate and those of the solvent either in the forward reaction direction (using dihydroxyacetone phosphate as substrate) or in the reverse direction (using glyceraldehyde phosphate as substrate). However, the specific radioactivity of the product glyceraldehyde phosphate formed in the forward reaction is 31% that of the solvent, while that of the product dihydroxyacetone phosphate formed in the reverse reaction is 24% that of the solvent. The deuterium kinetic isotope effects observed with the mutant isomerase using [1(R)-2H]dihydroxyacetone phosphate and [2-2H]glyceraldehyde 3-phosphate are 2.15 +/- 0.04 and 2.4 +/- 0.1, respectively. These results lead to the conclusion that substitution of Gln for His-95 so impairs the ability of the enzyme to stabilize the reaction intermediate that there is a change in the pathways of proton transfer mediated by the mutant enzyme. The data allow us more closely to define the role of His-95 in the reaction catalyzed by the wild-type enzyme, while forcing us to be alert to subtle changes in mechanistic pathways when mutant enzymes are generated.     

Reaction energetics of a mutant triosephosphate isomerase in which the active-site glutamate has been changed to aspartate

The essential catalytic base at the active site of the glycolytic enzyme triosephosphate isomerase is the carboxylate group of Glu-165, which directly abstracts either the 1-pro-R proton of dihydroxyacetone phosphate or the 2-proton of (R)-glyceraldehyde 3-phosphate to yield the cis-enediol intermediate. Using the methods of site-directed mutagenesis, we have replaced Glu-165 by Asp. The three enzymes chicken isomerase from chicken muscle, wild-type chicken isomerase expressed in Escherichia coli, and mutant (Glu-165 to Asp) chicken isomerase expressed in E. coli have each been purified to homogeneity. The specific catalytic activities of the two wild-type isomerases are identical, while the specific activity of the mutant enzyme is reduced by a factor of about 1000. The observed kinetic differences do not derive from a change in mechanism in which the aspartate of the mutant enzyme acts as a general base through an intervening water molecule, because the D2O solvent isotope effects and the stoichiometries of inactivation with bromohydroxyacetone phosphate are identical for the wild-type and mutant enzymes. Using the range of isotopic experiments that were used to delineate the free-energy profile of the wild-type chicken enzyme, we here derive the complete energetics of the reaction catalyzed by the mutant protein. Comparison of the reaction energetics for the wild-type and mutant isomerases shows that only the free energies of the transition states for the two enolization steps have been seriously affected. Each of the proton abstraction steps is about 1000-fold slower in the mutant enzyme. Evidently, the excision of a methylene group from the side chain of the essential glutamate has little effect on the free energies of the intermediate states but dramatically reduces the stabilities of the transition states for the chemical steps in the catalyzed reaction.     

Hydron transfer catalyzed by triosephosphate isomerase. Products of isomerization of (R)-glyceraldehyde 3-phosphate in D2O

The product distributions for the reactions of (R)-glyceraldehyde 3-phosphate (GAP) in D(2)O at pD 7.5-7.9 catalyzed by triosephosphate isomerase (TIM) from chicken and rabbit muscle were determined by (1)H NMR spectroscopy. Three products were observed from the reactions catalyzed by TIM: dihydroxyacetone phosphate (DHAP) from isomerization with intramolecular transfer of hydrogen (49% of the enzymatic products), [1(R)-(2)H]-DHAP from isomerization with incorporation of deuterium from D(2)O into C-1 of DHAP (31% of the enzymatic products), and [2(R)-(2)H]-GAP from incorporation of deuterium from D(2)O into C-2 of GAP (21% of the enzymatic products). The similar yields of [1(R)-(2)H]-DHAP and [2(R)-(2)H]-GAP from partitioning of the enzyme-bound enediol(ate) intermediate between hydron transfer to C-1 and C-2 is consistent with earlier results, which showed that there are similar barriers for conversion of this intermediate to the alpha-hydroxy ketone and aldehyde products (Knowles, J. R., and Albery, W. J. (1977) Acc. Chem. Res. 10, 105-111). However, the observation that the TIM-catalyzed isomerization of GAP in D(2)O proceeds with 49% intramolecular transfer of the (1)H label from substrate to product DHAP stands in sharp contrast with the 相似文献   

Hydron transfer catalyzed by triosephosphate isomerase. Products of isomerization of dihydroxyacetone phosphate in D2O

The product distributions for the reactions of dihydroxyacetone phosphate (DHAP) in D(2)O at pD 7.9 catalyzed by triosephosphate isomerase (TIM) from chicken and rabbit muscle were determined by (1)H NMR spectroscopy using glyceraldehyde 3-phosphate dehydrogenase to trap the first-formed products of the thermodynamically unfavorable isomerization reaction, (R)-glyceraldehyde 3-phosphate (GAP) and [2(R)-(2)H]-GAP (d-GAP). Three products were observed from the reactions catalyzed by TIM: GAP from isomerization with intramolecular transfer of hydrogen (18% of the enzymatic products), d-GAP from isomerization with incorporation of deuterium from D(2)O into C-2 of GAP (43% of the enzymatic products), and [1(R)-(2)H]-DHAP (d-DHAP) from incorporation of deuterium from D(2)O into C-1 of DHAP (40% of the enzymatic products). The ratios of the yields of the deuterium-labeled products d-DHAP and d-GAP from partitioning of the intermediate of the TIM-catalyzed reactions of GAP and DHAP in D(2)O are 1.48 and 0.93, respectively. This provides evidence that the reaction of these two substrates does not proceed through a single, common, reaction intermediate but, rather, through distinct intermediates that differ in the bonding and arrangement of catalytic residues at the enediolate O-1 and O-2 oxyanions formed on deprotonation of GAP and DHAP, respectively.     

Kinetic parameters for the elimination reaction catalyzed by triosephosphate isomerase and an estimation of the reaction's physiological significance

Kinetic parameters for triosephosphate isomerase catalysis of the elimination reaction of an equilibrium mixture of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (DGAP) to form methylglyoxal and phosphate ion are reported for the enzyme from rabbit muscle. Pseudo-first-order rate constants for the disappearance of substrate (kelim) were determined for reactions at [Enzyme] much greater than [Substrate]. The second-order rate constant kEnz = 10.1 M-1 s-1 was determined from a plot of kelim against enzyme concentration. The kinetic parameters, determined from a steady-state kinetic analysis at [Substrate] much greater than [Enzyme], are kcat = 0.011 s-1, Km = 0.76 mM, and kcat/Km = 14 M-1 s-1. The estimated rate-constant ratio for partitioning of the enzyme-bound intermediate between protonation at carbon 2 and elimination, 1,000,000, is much larger than the ratio of 6.5 determined for the reaction of the enediolate phosphate in a loose complex with quinuclidinonium cation, a small buffer catalyst. There is a 10(5)-10(8)-fold decrease in the rate constant for the elimination reaction of the enediolate phosphate when this species binds to triosephosphate isomerase. The kinetic parameters for the elimination reaction catalyzed by the native triosephosphate isomerase and for the reaction catalyzed by a mutant form of the enzyme, which is missing a segment that forms hydrogen bonds with the phosphate group of substrate [Pompliano, D. L., Peyman, A., & Knowles, J. R. (1990) Biochemistry 29, 3186-3194] are similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic properties of triose-phosphate isomerase from Trypanosoma brucei brucei. A comparison with the rabbit muscle and yeast enzymes

The kinetic properties of Trypanosoma brucei brucei triose-phosphate isomerase are compared with those of the commercially available rabbit muscle and yeast enzymes and with published data on the chicken muscle enzyme. With glyceraldehyde 3-phosphate as substrate Km = 0.25 +/- 0.05 mM and kcat = 3.7 X 10(5) min-1. With dihydroxyacetone phosphate as substrate Km = 1.2 +/- 0.1 mM and kcat = 6.5 X 10(4) min-1. The pH dependence of Km and Vmax at 0.1 M ionic strength is in agreement with the results published for the yeast and chicken muscle enzymes. At ionic strength below 0.05 M the effect of a charged group specific for the trypanosomal enzyme and absent from the yeast and rabbit muscle enzymes becomes detectable. This effect significantly increases Km whereas Vmax becomes slightly higher. Trypanosomal triose-phosphate isomerase is inhibited by sulphate, phosphate and arsenate ions, by 2-phosphoglycolate and a number of documented inhibitors in the same concentration range as are the other triose-phosphate isomerases. The trypanocidal drug, Suramin inhibits T. brucei and rabbit muscle triose-phosphate isomerase to the same extent while leaving the yeast enzyme relatively unaffected.     

Ribulose-1,5-bisphosphate carboxylase: enzyme-catalyzed appearance of solvent tritium at carbon 3 of ribulose 1,5-bisphosphate reisolated after partial reaction

When ribulose 1,5-bisphosphate is allowed to react with carbon dioxide in tritiated water in the carboxylation reaction catalyzed by ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum, the ribulose 1,5-bisphosphate reisolated after partial reaction is found to be labeled. The specific radioactivity of the remaining substrate pool rises during the course of the reaction. Experiments in deuterium oxide show that the isotopic label resides on carbon 3. Earlier failures to detect this exchange process probably derive from the use of enzyme that was, in the absence of carbon dioxide, inactive. The present results provide direct evidence for the intermediacy of the enediol between C-2 and C-3 of ribulose 1,5-bisphosphate and show that the enolization step is at least partially rate limiting in the overall carboxylase reaction. The specific radioactivity of the product 3-phospho-D-glycerate remains constant throughout the course of the reaction at about one-sixth that of the solvent. This strengthens the argument against the involvement of "sticky" protons in the reaction.     

Role of mono- and divalent metal cations in the catalysis by yeast aldolase

The rate of deuterium exchange between [1-(S)-2H]dihydroxyacetone 3-phosphate and the solvent catalyzed by native and metal-substituted yeast aldolases has been measured. In the presence of 0.1 M potassium acetate at 15 degrees C, pH 7.3, the deuterium exchange reaction catalyzed by native yeast aldolase has a kcat of 95 s-1. In contrast to the 7-fold activity enhancement by 0.1 M potassium ion (relative to 0.1 M sodium ion) of the cleavage of D-fructose 1,6-bisphosphate catalyzed by native yeast aldolase, a negligible (1.1-fold) activation by 0.1 M potassium ion is observed in the rate of dedeuteration of [1(S)-2H]dihydroxyacetone 3-phosphate. The order of reactivity of the yeast metalloaldolases in the deuterium exchange roughly parallels that seen in the fructose bisphosphate cleavage reaction. These findings suggest that the carbonyl groups of enzyme-bound D-fructose 1,6-bisphosphate and dihydroxyacetone phosphate are both polarized by the active site divalent metal cation. A mechanistic formulation consistent with the results of this and the previous paper is presented.     

Specificity and kinetics of triose phosphate isomerase from chicken muscle

The isolation of crystalline triose phosphate isomerase from chicken breast muscle is described. The values of k(cat.) and K(m) for the reaction in each direction were determined from experiments over wide substrate-concentration ranges, and the reactions were shown to obey simple Michaelis-Menten kinetics. With d-glyceraldehyde 3-phosphate as substrate, k(cat.) is 2.56x10(5)min(-1) and K(m) is 0.47mm; with dihydroxyacetone phosphate as substrate, k(cat.) is 2.59x10(4)min(-1) and K(m) is 0.97mm. The enzyme-catalysed exchange of the methyl hydrogen atoms of the ;virtual substrate' monohydroxyacetone phosphate with solvent (2)H(2)O or (3)H(2)O was shown. This exchange is about 10(4)-fold slower than the corresponding exchange of the C-3 hydrogen of dihydroxyacetone phosphate. The other deoxy substrate, 3-hydroxypropionaldehyde phosphate, was synthesized, but is too unstable in aqueous solution for analogous proton-exchange reactions to be studied.     

Mechanism of the reaction catalyzed by mandelate racemase. 1. Chemical and kinetic evidence for a two-base mechanism

The fate of the alpha-hydrogen of mandelate in the reaction catalyzed by mandelate racemase has been investigated by a mass spectroscopic method. The method entails the incubation of (R)- or (S)-[alpha-1H]mandelate in buffered D2O to a low extent of turnover (about 5-8%), esterification of the resulting mixture of mandelates with diazomethane, derivatization of the methyl esters with a chiral derivatizing agent, and quantitation of the isotope content of the alpha-hydrogen of both substrate and product by gas chromatography/mass spectrometric analysis. No significant substrate-derived alpha-protium was found in the product for racemization in either direction. In addition, in the (R) to (S) direction almost no exchange (less than or equal to 0.4%) of the alpha-hydrogen in the remaining (R) substrate pool occurred, but in the (S) to (R) direction 3.5-5.1% exchange of the alpha-hydrogen in the remaining substrate (after 5.1-7.2% net turnover) was found. Qualitatively similar results were obtained in the (S) to (R) direction in H2O when (S)-[alpha-2H]mandelate was used as substrate. In other experiments, an overshoot in the progress curve was observed when the racemization of either enantiomer of [alpha-1H]mandelate in D2O was monitored by following the change in ellipticity of the reaction mixture; the magnitude of the overshoot was greater in the (R) to (S) than in the (S) to (R) direction. All of the available data indicate that the reaction catalyzed by mandelate racemase proceeds by a two-base mechanism, in contrast to earlier proposals.     

Proton transfer in methylmalonyl-CoA epimerase from Propionibacterium shermanii. Studies with specifically tritiated (2R)-methylmalonyl-CoA as substrate.

(2R)-Methyl[2-3H]malonyl-CoA was used as the substrate for methylmalonyl-CoA epimerase from Propionibacterium shermanii, under conditions where the (2S)-methylmalonyl-CoA product was removed enzymically as fast as it was formed, and the fate of the label was monitored at different extents of reaction. Very little, if any, tritium is found attached to the C-2 position in the (2S)-epimer product (isolated as propionyl-CoA). Evidently, the hydrogen atom of the new C-H bond in the product is essentially solvent-derived. The rate of tritium release into the solvent is lower than the rate of product formation, and shows a primary kinetic tritium-isotope effect on kcat./Km of 2.3 +/- 0.1. The specific radioactivity of the remaining substrate rises slowly during the epimerase-catalysed reaction, and this provides an independent estimate of the primary kinetic tritium-isotope effect on kcat./Km of 1.6 +/- 0.5. These results, taken together, indicate that the mechanistic pathway of the epimerase-catalysed reaction resembles that established for proline racemase [Cardinale & Abeles, (1968) Biochemistry 7, 3970-3978], in which two enzyme bases are involved in catalysis. One base removes the proton from the substrate, the second provides the new proton, and there is no fast isotopic exchange between enzyme-bound intermediates and solvent protons.     

Proton transfer in methylmalonyl-CoA epimerase from Propionibacterium shermanii. The reaction of (2R)-methylmalonyl-CoA in tritiated water.

The reaction catalysed by methylmalonyl-CoA epimerase from Propionibacterium shermanii was studied in tritiated water, in the direction with (2R)-methylmalonyl-CoA as substrate, under 'irreversible' conditions. After partial reaction, even when most of the substrate had been converted into product (isolated as propionyl-CoA) essentially no solvent tritium appeared in residual (2R)-methylmalonyl-CoA. The product, however, did contain tritium, and the specific radioactivity of the (2S)-epimer was deduced to be 0.33 times that of the solvent. These results provide further support for the mechanism proposed for the epimerase-catalysed reaction in the accompanying paper [Leadlay & Fuller (1983) Biochem. J. 213, 635-642], in which two enzyme bases act respectively as proton donor and acceptor. The observed low discrimination against solvent tritium entering the product can be accounted for by a mechanism in which the release of product is slow, and the re-protonation step on the enzyme is reversible, without leading to isotopic exchange with the solvent.     

Enzyme relaxation in the reaction catalyzed by triosephosphate isomerase: detection and kinetic characterization of two unliganded forms of the enzyme

Triosephosphate isomerase has been shown to exist in two unliganded forms, one of which binds and isomerizes (R)-glyceraldehyde 3-phosphate and the other of which binds and isomerizes dihydroxyacetone 3-phosphate. The tracer perturbation method of Britton demonstrates the kinetic significance of the interconversion of these two enzyme forms at high substrate concentrations and yields a rate constant of about 10(6) s-1 for the interconversion. Although the molecular nature of the two forms of unliganded enzyme is not defined by these experiments, a shuffling of protons among active site residues, or a protein conformational change, or both, may be involved. This study, coupled with the known rate constants for the substrate-handling steps of triosephosphate isomerase catalysis, completes the kinetic characterization of the catalytic cycle for this enzyme.     

The uncatalyzed rates of enolization of dihydroxyacetone phoshate and of glyceraldehyde 3-phosphate in neutral aqueous solution. The quantitative assessment of the effectiveness of an enzyme catalyst.

By a combination of methods involving enzyme-catalyzed reactions and classical iodination techniques it has been possible to obtain all the relevant rate constants for the uncatalyzed interconversion of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate via their common enediol intermediate. These rate constants are compared with those for the individual steps of the triosephosphate isomerase catalyzed reaction, and a quantitative picture of the effectiveness of the enzyme as a catalyst has been delineated. It is apparent that the enzyme increases the enolization rate of dihydroxyacetone phosphate by a factor of more than 10(9) over that of the uncatalyzed reaction.     

Ab initio models for receptor-ligand interactions in proteins. 4. Model assembly study of the catalytic mechanism of triosephosphate isomerase

The catalytic mechanism of triosephosphate isomerase (TIM) was investigated with ab initio quantum mechanical calculations. Electrostatic interactions between the quantum mechanical active site and the protein and solvent environment were modeled using the finite difference Poission-Boltzman method. The complexes of TIM with the substrate dihydroxyacetone phosphate (DHAP), five possible intermediates and the product glyceraldehyde-3-phosphate (GAP) were optimized in the active-site model at the 3-21G^(*) level and energy profile for the proton abstraction from DHAP by the active-site Glu167 was calculated at the MP2/3-21G^(*)//3-21G^(*) level. Calculated energetics of the enzyme reaction were found to be in reasonable agreement with the experimental findings. Calculations revealed that an enediol of the substrate is a probable intermediate in the enzyme reaction. It was suggested that the proton abstracted from the substrate by the active-site glutamate goes to the carbonyl oxygen of the substrate producing enediol intermediate either directly or after it is exchanged with solvent. © 1996 Wiley-Liss, Inc.     

Critical ionization states in the reaction catalyzed by triosephosphate isomerase.

To allow the detailed interpretation of the pH dependences of the steady-state parameters for the reaction catalyzed by triosephosphate isomerase, three kinds of experiments have been performed. First, the value of kcat/Km for enzyme-catalyzed isomerization of the phosphonate analogue of D-glyceraldehyde 3-phosphate (2-hydroxy-4-phosphonobutyraldehyde) has been shown to titrate with an apparent pKa of 7.5, which is close to the phosphonate's second ionization constant. Secondly, the sulfate ester analogue of dihydroxyacetone phosphate (dihydroxyacetone sulfate), which exists only as a monoanion over the pH range of interest, has been shown not to bind detectably to the enzyme. Thirdly, an isotopic discrimination experiment at pH 5.2 has been compared with a similar investigation at pH 7.6. The results together demonstrate that both enzyme and substrate ionizations control the reaction rate in the pH range 5 to 8.     

GLYCEROL KINASE AND DIHYDROXYACETONE KINASE IN RAT BRAIN

—The enzymatic phosphorylation of glycerol and dihydroxyacetone by ATP to sn-glycerol-3-phosphate and dihydroxyacetone phosphate respectively in various subcellular fractions of rat brain was studied. A sensitive radiochemical assay was used where the labelled phosphorylated products were separated from the radioactive substrates by high voltage paper electrophoresis and the radioactivity in these compounds determined. Using this assay the glycerol kinase (EC 2.7.1.30) activity was found to be associated with the mitochondrial fraction of the brain. Under optimum conditions 2.45 nmol of glycerol was phosphorylated/min per mg of protein. The K_m for glycerol was 70 μm at pH 7. This mitochondrial enzyme, like other glycerol kinases from different sources, also phosphorylated dihydroxyacetone. Under optimum conditions 1.7 nmol of dihydroxyacetone phosphate was formed/min per mg of mitochondrial protein. The K_m for dihydroxyacetone was 0.6 mm . Glycerol kinase activity was also present in the cytoplasm of brain. However, the specific activity of this enzyme in cytosol is about 15% of the mitochondrial glycerol kinase. Compared to glycerol, dihydroxyacetone was phosphorylated by ATP in cytoplasm at a much higher rate. The pH optimum for this soluble dihydroxyacetone kinase was much lower (pH 6.5) than that of the soluble or mitochondrial glycerol kinase (pH 10.0). Using ammonium sulfate, brain cytoplasm was fractionated to yield a fraction in which the dihydroxyacetone kinase was enriched 2–3 fold with no glycerol kinase activity. Under optimum conditions 1.0 nmol of dihydroxyacetone was phosphorylated/min per mg protein. The K_m for dihydroxyacetone was 60 μm . This cytosol fraction was also found to phosphorylate d -glyceraldehyde and l -glyceraldehyde at a rate of 30–40% to that of the dihydroxyacetone phosphorylation. The properties and the possible metabolic role of these enzymes in brain are discussed.     

The existence of an electrophilic component in the reaction catalysed by triose phosphate isomerase (Short Communication)

In the presence of triose phosphate isomerase, the substrate dihydroxyacetone phosphate is reduced stereoselectively by NaBH(4). The reduction of enzyme-bound substrate is almost completely or completely stereoselective and occurs about one order of magnitude faster than that in free solution. This acceleration implies a polarization of the carbonyl group when dihydroxyacetone phosphate is bound.     

Electrophilic catalysis in triosephosphate isomerase: the role of histidine-95

Electrophilic catalysis by histidine-95 in triosephosphate isomerase has been probed by using Fourier transform infrared spectroscopy and X-ray crystallography. The carbonyl stretching frequency of dihydroxyacetone phosphate bound to the wild-type enzyme is known to be 19 cm-1 lower (at 1713 cm-1) than that of dihydroxyacetone phosphate free in solution (at 1732 cm-1), and this decrease in stretching frequency has been ascribed to an enzymic electrophile that polarizes the substrate carbonyl group toward the transition state for the enolization. Infrared spectra of substrate bound to two site-directed mutants of yeast triosephosphate isomerase in which histidine-95 has been changed to glutamine or to asparagine show unperturbed carbonyl stretching frequencies between 1732 and 1742 cm-1. The lack of carbonyl polarization when histidine-95 is removed suggests that histidine-95 is indeed the catalytic electrophile, at least for dihydroxyacetone phosphate. Kinetic studies of the glutamine mutant (H95Q) have shown that the enzyme follows a subtly different mechanism of proton transfers involving only a single acid-base catalytic group. These findings suggest an additional role for histidine-95 as a general acid-base catalyst in the wild-type enzyme. The X-ray crystal structure of the H95Q mutant with an intermediate analogue, phosphoglycolohydroxamate, bound at the active site has been solved to 2.8-A resolution, and this structure clearly implicates glutamate-165, the catalytic base in the wild-type isomerase, as the sole acid-base catalyst for the mutant enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzymes involved in the formation of glycerol 3-phosphate and the by-products dihydroxyacetone and glycerol in Zymomonas mobilis

Abstract In Zymomonas mobilis a novel pathway for the formation of glycerol 3-phosphate was identified by enzymatic studies and nuclear magnetic resonance spectroscopy. This pathway branches off from the Entner-Doudoroff pathway at the intermediate glyceraldehyde 3-phosphate and proceedes via dihydroxyacetone phosphate, dihydroxyacetone, glycerol to glycerol 3-phosphate. The reaction sequence is catalyzed by the enzymes triosephosphate isomerase (0.4 U (mg protein)⁻¹), dihydroxyacetone phosphatase (0.31 U (mg protein)⁻¹), dihydroxyacetone reductase (0.25 U (mg protein)⁻¹), and glycerokinase (0.08 mU (mg protein)⁻¹), respectively. The action of a postulated aldolase catalyzing the cleavage of fructose 6-phosphate to dihydroxyacetone and glyceraldehyde 3-phosphate could be excluded.