Anomerization rates and enzyme specificity for biologically important sugars and sugar phosphates

Schray, Keith J.; Benkovic, Stephen J.

doi:10.1021/ar50124a002

Cited by 35 publications

(32 citation statements)

References 36 publications

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“…Therefore, our results with the 4-deoxy analogue 1 are in accordance with the hypothesis that the aldehyde form of AraSP, the minor form in solution, functions as the true substrate of the enzyme. There are other cases of enzymes that show a preference for minor isomeric forms of sugar substrates (Schray and Benkovic, 1978). Aldolase, glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase, use the aldehyde form of glyceraldehyde-3-phosphate exclusively.…”

Section: Discussionmentioning

confidence: 99%

Mechanistic studies of 3‐deoxy‐d‐manno‐2‐octulosonate‐8‐phosphate synthase from Escherichia coli

Kohen¹,

Jakob²,

Baasov³

1992

European Journal of Biochemistry

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The anomeric specificity and the steady-state kinetic mechanism of homogeneous 3-deoxy-~-manno-2-octulosonate-8-phosphate (KD08P) synthase were investigated. The open-chain 4-deoxy analogue of arabinose-5-phosphate (AraSP), which is structurally prohibited from undergoing ring closure, was synthesized and tested as a substrate for the synthase. It was found that the analogue functions as a substrate with a similar k,,, value to that of the original substrate. The k,,,/Km value for the natural substrate is seven-times greater than that of the 4-deoxy analogue. However, taking into account the 9.5% and approximately 1% concentrations of the aldehyde forms of the 4-deoxy analogue and AraSP in solution, then the 'true' K,,, values must be in the range 31.5 pM and 0.26 pM, respectively, requiring about a 3 kcal/mol contribution to the binding energy by the 4-hydroxyl group of AraSP. The data provides evidence that the enzyme acts upon the acyclic form of the natural substrate.The steady-state kinetic study of KD08P synthase was analyzed via inhibition using the products KD08P and inorganic phosphate, and ~-ribose-5-phosphate as a dead-end inhibitor. First, intersecting lines in double-reciprocal plots of initial-velocity data at substrate concentrations in the micromolar range suggest a sequential mechanism for the enzyme-catalyzed reaction. The inhibition by ~-ribose-5-phosphate is competitive for Ara5P and uncompetitive for phosphoenolpyruvate (Ppyruvate). These inhibition patterns are consistent with the model wherein P-pyruvate binding precedes that of Ara5P binding. Furthermore, this order of substrate binding was supported by the observations that KD08P is a competitive inhibitor for P-pyruvate binding, supporting the concept that KD08P and P-pyruvate bind to the same enzyme form, and noncompetitively with respect to AraSP. In addition, the inhibition by inorganic phosphate is noncompetitive with respect to both Ppyruvate and AraSP, suggesting an apparent ordered release of products such that Pi first, followed by KD08P. In conclusion, these data suggest a steady-state kinetic mechanism for KD08P synthase where P-pyruvate binding precedes that of AraSP, followed by the ordered release of inorganic phosphate and KD08P.3-Deoxy-~-manno-2-octulosonate-8-phosphate (KD08P) synthase is a key enzyme which controls the carbon flow in the biosynthetic formation of 3deoxy-~-munno-2-octulosonate (KDO; Unger, 1981; Inouye, 1979), a specific constituent of the lipopolysaccharide of most Gram-negative bacteria . Since the biosynthesis of lipopolysaccharide is unique to Gram-negative bacteria and required by them for growth and virulence, several groups have pursued inhibition of KDO metabolism as a strategy for the development of novel antiinfective agents (Hammond et al., 1987; Goldman et al., 1987). KD08P synthase catalyzes the condensation of D-arabinose 5-phosphate (AraSP) with phosphoenolpyruvate (Ppyruvate) to produce the unusua1 eight-carbon saccharide KD08P and inorganic phosphate (Pi;Ray, 1980). A similar reaction b...

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Section: Discussionmentioning

confidence: 99%

Mechanistic studies of 3‐deoxy‐d‐manno‐2‐octulosonate‐8‐phosphate synthase from Escherichia coli

Kohen¹,

Jakob²,

Baasov³

1992

European Journal of Biochemistry

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“…b If the substrate for the chloroplast enzyme is the d anomer as it is for other glucose 6-P dehydrogenases (19), the Km (glucose 6-P) values are 220 uMm for the dark form and 87 AM for the light form of the enzyme. c Note that this value is an average value in these experiments.…”

Section: Dark Formmentioning

confidence: 99%

Changing Activity of Glucose-6-Phosphate Dehydrogenase from Pea Chloroplasts during Photosynthetic Induction

Yuan

Anderson²

1987

Plant Physiol.

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Light inactivation of glucose 6-phosphate dehydrogenase is rapid and occurs before photosynthetic 02 evolution is measureable in intact chloroplasts. Likewise, dark activation is rapid. The major light induced change in the kinetic parameters of glucose 6-phosphate dehydrogenase is in maximal velocity.The first enzyme of the oxidative pentose phosphate pathway, glucose 6-P dehydrogenase (EC 1.1.1.49), is inactivated when pea chloroplasts are irradiated (3). This light inactivation probably prevents operation of a futile cycle involving glucose 6-P, NADPH, and oxidative and reductive pentose phosphate pathway enzymes. We have now examined the kinetics of light inactivation of glucose 6-P dehydrogenase in intact chloroplasts during photosynthetic induction and the kinetic parameters of the active (dark) and less active (light) forms of the enzyme.Fickenscher and Scheibe (9) have partially purified cytosolic glucose 6-P dehydrogenase from peas. Unlike the purified chloroplastic enzyme (20), the cytosolic enzyme was not DTT or DTT/thioredoxin sensitive. On the basis ofthis lack of sensitivity to DTT and the results of antibody experiments with extracts from leaves which had been illuminated, Fickenscher and Scheibe concluded that the cytosolic enzyme was not light inactivated. But in their antibody experiments the error in activity determinations for the cytosolic enzyme from darkened and illuminated leaves was 18 and 23%, respectively (Fig. 3 in Ref. 9). Since errors are additive, the error in the determination of light inactivation was then the same as the expected change in activity if the cytosolic enzyme were 40% inactivated by light. Because previous work in this laboratory (4-6) indicated that the cytosolic enzyme was light and DTT inactivated, we have reinvestigated the DTT-dependent inactivation of the enzyme in whole leaf extracts. MATERIALS AND METHODSPhotosynthetic 02 Evolution and Determination of Kinetic Parameters. Chloroplasts were isolated from pea (Pisum sativum L. var Little Marvel) seedlings and photosynthetic 02 evolution assayed as described by Marques and Anderson (15) except that the chloroplast concentration was increased. Where activity with time of illumination was followed, the chloroplast content in the

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“…[14] Under physiological conditions, GlcN6P equilibrates between an acyclic form (1 b) and two cyclic b-anomer (1 a) and a-anomer (1 c) forms (Figure 1 b). [15] The relative ratio of 1 a and 1 c in solution is 60:40 at 25 8C as determined by 1 H NMR spectroscopy in D 2 O (data not shown), with less than 1 % in the acyclic form. [15] Each conformer could exhibit differences in RNA binding affinity and ribozyme activity similar to that observed for the GlmS protein.…”

mentioning

confidence: 92%

“…[15] The relative ratio of 1 a and 1 c in solution is 60:40 at 25 8C as determined by 1 H NMR spectroscopy in D 2 O (data not shown), with less than 1 % in the acyclic form. [15] Each conformer could exhibit differences in RNA binding affinity and ribozyme activity similar to that observed for the GlmS protein. [16] Small molecules such as serinol and ethanolamine promote ribozyme activity, although they are orders of magnitude less effective than GlcN6P.…”

mentioning

confidence: 92%

Characteristics of Ligand Recognition by a glmS Self‐Cleaving Ribozyme

Lim¹,

Grove²,

Roth³

et al. 2006

Angew Chem Int Ed

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The glmS ribozyme [1][2][3][4][5][6][7] from Bacillus cereus is a representative of a unique riboswitch class [8,9] whose members undergo selfcleavage with accelerated rate constants when bound to glucosamine-6-phosphate (GlcN6P). These metabolite-sensing ribozymes are found in numerous Gram-positive bacteria, where they control expression of the glmS gene. The glmS gene product (glutamine:fructose-6-phosphate amidotransferase) generates GlcN6P, [10,11] which binds to the ribozyme and triggers self-cleavage by internal phosphoester transfer.[1]The ribozyme is embedded within the 5' untranslated region (UTR) of the glmS messenger RNA and self-cleavage prevents production of GlmS protein, thereby decreasing the concentration of GlcN6P. The combination of molecular

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Anomerization rates and enzyme specificity for biologically important sugars and sugar phosphates

Cited by 35 publications

References 36 publications

Mechanistic studies of 3‐deoxy‐d‐manno‐2‐octulosonate‐8‐phosphate synthase from Escherichia coli

Mechanistic studies of 3‐deoxy‐d‐manno‐2‐octulosonate‐8‐phosphate synthase from Escherichia coli

Changing Activity of Glucose-6-Phosphate Dehydrogenase from Pea Chloroplasts during Photosynthetic Induction

Characteristics of Ligand Recognition by a glmS Self‐Cleaving Ribozyme

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