Incubations of testis myo-inositol-1-phosphate synthase with D-(5-18O)glucose 6-phosphate and with H218O show no evidence of Schiff base formation.

Sherman, William R.; Rasheed, A.; Mauck, Linda; Wiecko, Jacek

doi:10.1016/s0021-9258(17)40075-5

Cited by 24 publications

(9 citation statements)

References 29 publications

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“…After the incubation period the samples were dephosphorylated with Escherichia coli alkaline phosphatase and analyzed for glucose by GC-MS using the acetate-butaneboronate derivative of glucose (Sherman et al, 1977). No glucose formation was observed in any of the samples.…”

Section: Resultsmentioning

confidence: 99%

“…Methods. The general procedures including sources of reagents, methods of preparing isotopically labeled dglucose-6-P, gas chromatographic and gas chromatographymass spectrometry techniques, derivatization, and enzyme assay procedures are described in detail in Sherman et al (1977).…”

Section: Methodsmentioning

confidence: 99%

“…L-myoinositol-1-P D-glucose 6-phosphate and the mechanism of the transformation seems to be similar, if not identical, regardless of which of the many sources of the enzyme have been studied [for a brief review see Sherman et al (1977)]. The M1P synthase is a complex enzyme combining the activities of an NAD+-dependent oxidoreductase with an aldolase.…”

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confidence: 99%

“…The same hydrogen (Sherman et al, 1969) is then returned to another intermediate (lmyo-inosose-2-l-P; Scheme I) to give l-MIP which is acted on by a specific phosphatase to give the final product (Eisenberg, 1967). The mammalian M1P synthases which have been studied are unusual aldolases in being neither Schiff base forming enzymes nor divalent metal requiring ones (Sherman et al, 1977). The proposed mechanism of the MlP synthase is outlined in Scheme I.…”

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confidence: 99%

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L-myo-Inositol-1-phosphate synthase from bovine testis: purification to homogeneity and partial characterization

Mauck¹,

Wong²,

Sherman³

1980

Biochemistry

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L-myo-Inositol-1-phosphate synthase has been purified to homogeneity from bovine testis by (NH4)2SO4 precipitation on Celite followed by reverse (NH4)2SO4 gradient elution, DEAE chromatography, gel filtration, and hydroxylapatite chromatography. The enzyme is then pure by the criteria of elution profile from the hydroxylapatite, electrophoresis, and sedimentation properties. We find no overall (gluconeogenic) reversibility of the enzyme using 6 mM DL-myo-inositol-1-P. The first three steps of the reaction are reversible as determined by uptake of isotope from a D2O incubation medium into the 6 position of D-glucose-6-P. Thus, substrate binding, dehydrogenation, and proton removal prior to the aldol cyclization are all reversible steps. The enzyme is less than 5% NAD+ independent and is not inhibited by substrate or product (5 mM D-glucose-6-P or 0.8 mM DL-myo-inositol-1-P). The enzyme is twofold stimulated by either 50 mM NH4+ or 50 mM K+; the activation by these ions is not additive. Sodium ions inhibit the enzyme by 78% at 153 mM. The effect of sodium and potassium is not on the Km of D-glucose-6-P but on Vmax. We propose that K+ activates the enzyme by stabilizing a carbanion intermediate. Ethanol stimulates the enzyme 2-fold and 2.5-fold with added K+. The effect of ethanol appears to be via lowering of the D-glucose-6-P Km. In the presence of ethanol the effect of salt on Vmax disappears.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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L-myo-Inositol-1-phosphate synthase from bovine testis: purification to homogeneity and partial characterization

Mauck¹,

Wong²,

Sherman³

1980

Biochemistry

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“…The eukaryotic enzymes are >550 residues, whereas the bacterial IPS homologues [identified by sequence alignment of a 150-residue conserved domain in the Cterminus of all IPS (26)] appear to be smaller with 350-420 residues. The catalytic mechanism, first proposed by Loewus and Kelly (1) and supported by others (1,(4)(5)(6), involves oxidation of substrate glucose-6-phosphate to 5-keto-(D)-glucose-6-phosphate, cyclization to myo-inosose-2 1-phosphate [which is also a competitive inhibitor of IPS (27)], and reduction to (L)-myo-inositol-1-phosphate (Scheme 1). Since the two keto intermediates have not been detected directly in studies of known IPS enzymes, it has been assumed that they are tightly bound to the enzyme.…”

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Inositol-1-phosphate Synthase fromArchaeoglobus fulgidusIs a Class II Aldolase

et al. 2000

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A gene putatively identified as the Archaeoglobus fulgidus inositol-1-phosphate synthase (IPS) gene was overexpressed to high level (about 30-40% of total soluble cellular proteins) in Escherichia coli. The recombinant protein was purified to homogeneity by heat treatment followed by two column chromatographic steps. The native enzyme was a tetramer of 168 +/- 4 kDa (subunit molecular mass of 44 kDa). At 90 degrees C the K(m) values for glucose-6-phosphate and NAD(+) were estimated as 0.12 +/- 0.04 mM and 5.1 +/- 0.9 microM, respectively. Use of (D)-[5-(13)C]glucose-6-phosphate as a substrate confirmed that the stereochemistry of the product of the IPS reaction was L-myo-inositol-1-phosphate. This archaeal enzyme, with the highest activity at its optimum growth temperature among all IPS reported (k(cat) = 9.6 +/- 0.4 s(-1) with an estimated activation energy of 69 kJ/mol), was extremely heat stable. However, the most unique feature of A. fulgidus IPS was that it absolutely required divalent metal ions for activity. Zn(2+) and Mn(2+) were the best activators with K(D) approximately 1 microM, while NH(4)(+) (a critical activator for all the other characterized IPS enzymes) had no effect on the enzyme. These properties suggested that this archaeal IPS was a class II aldolase. In support of this, stoichiometric reduction of NAD(+) to NADH could be followed spectrophotometrically when EDTA was present along with glucose-6-phosphate.

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The Structure and Mechanism of myo-Inositol-1-Phosphate Synthase

Geiger

Jin

Subcellular Biochemistry

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Abstract:The first and rate-limiting step in the biosynthesis of myo-inositol is the conversion of D-glucose 6-phosphate to 1L-myo-inositol 1-phosphate catalyzed by 1L-myo-inositol 1-phosphate synthase (MIP synthase). MIP synthase has been identified in a wide variety of organisms from bacteria to humans and is relatively well-conserved throughout evolution. It is probably homotetrameric in most if not all cases and always requires NAD ϩ as a cofactor, with NADH being reconverted to NAD ϩ in the catalytic cycle. This review focuses on the structure and mechanism of MIP synthase, with a particular emphasis on the mechanistic insights that have come from several recent structures of the enzyme. These include the structure of the enzyme from Saccharomyces cerevisiae, Archeoglobus fulgidus and Mycobacterium tuberculosis.

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Incubations of testis myo-inositol-1-phosphate synthase with D-(5-18O)glucose 6-phosphate and with H218O show no evidence of Schiff base formation.

Cited by 24 publications

References 29 publications

L-myo-Inositol-1-phosphate synthase from bovine testis: purification to homogeneity and partial characterization

L-myo-Inositol-1-phosphate synthase from bovine testis: purification to homogeneity and partial characterization

Inositol-1-phosphate Synthase fromArchaeoglobus fulgidusIs a Class II Aldolase

The Structure and Mechanism of myo-Inositol-1-Phosphate Synthase

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