Interactions of pig liver serine hydroxymethyltransferase with methyl tetrahydropteroylpolyglutamate inhibitors and with tetrahydropteroylpolyglutamate substrates

Matthews, Rowena G.; Ross, Jonathan; Baugh, Charles M.; Cook, Janine D.; Davis, Leodis

doi:10.1021/bi00535a019

Cited by 55 publications

(52 citation statements)

References 23 publications

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“…The orientation of PLP in the tetramer, as monitored by visible CD (Figure 1b), indicated that it was in the correct orientation for catalysis. The quinonoid spectral intermediate, commonly seen in the equilibrium mixture of SHMT, glycine and H % -folate, was shown to have an absorption coefficient of 50 000 M −" :cm −" in the case of rabbit liver [37] and pig liver [38] SHMTs. In the case of rSHMT the value was calculated to be 29 255 M −" :cm −" in presence of 500 µM PLP.…”

Section: Discussionmentioning

confidence: 99%

Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

et al. 1999

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Aspartate residues function as proton acceptors in catalysis and are involved in ionic interactions stabilizing subunit assembly. In an attempt to unravel the role of a conserved aspartate (D89) in sheep-liver tetrameric serine hydroxymethyltransferase (SHMT), it was converted into aspargine by site-directed mutagenesis. The purified D89N mutant enzyme had a lower specific activity compared with the wild-type enzyme. It was a mixture of dimers and tetramers with the proportion of tetramers increasing with an increase in the pyridoxal-5h-phosphate (PLP) concentration used during purification. The D89N mutant tetramer was as active as the wild-type enzyme and had similar kinetic and spectral properties in the presence of 500 µM PLP. The quinonoid spectral intermediate commonly seen in the case of SHMT was also seen in the case of D89N mutant tetramer, although the amount of intermediate formed was lower. Although the purified dimer exhibited visible absorbance at 425 nm, it had a negligible visible CD spectrum at 425 nm and was only 5 % active. The

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Section: Discussionmentioning

confidence: 99%

Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

et al. 1999

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“…The enzyme was purified to homogeneity using polyethyleneimine precipitation, DEAESepharose chromatography, and SP-Sepharose chromatography. The activity of purified PvSHMT was assayed at 25°C under anaerobic conditions by coupling its reaction with the reaction of His 6 -tagged MTHFR (5,21,22). In brief, a mixture of enzyme solution containing PvSHMT (1 M) and His 6 -tagged MTHFR (3 M) in 50 mM HEPES, pH 7.0, containing 0.5 mM EDTA, and 1 mM DTT was mixed with a substrate solution containing NADH (100 M), L-serine (2 mM), and H 4 folate (400 M) at 25°C under anaerobic conditions by using a stoppedflow spectrophotometer (TgK Scientific instruments, models SF-61DX2 or SF-61SX).…”

Section: Methodsmentioning

confidence: 99%

Kinetic Mechanism and the Rate-limiting Step of Plasmodium vivax Serine Hydroxymethyltransferase

Maenpuen

Amornwatcharapong

Krasatong

et al. 2015

Journal of Biological Chemistry

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Background: Plasmodium vivax serine hydroxymethyltransferase (PvSHMT) catalyzes formation of glycine from L-serine and tetrahydrofolate. Results: Results indicate that PvSHMT can bind to either substrate first. The rate constant of glycine formation is similar to k cat . Conclusion: PvSHMT reaction occurs via a random-order mechanism and glycine formation is the rate-limiting step. Significance: The data are useful for future investigation on inhibition of SHMT for antimalarial drug development.

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“…An increase in glutamate chain length from 1 to 6 increases the affinity of H 4 PteGlu n for several mammalian SHMTs by ϳ2 orders of magnitude, but results in only a 2-fold increase in affinity for Escherichia coli SHMT (22,23). Crystal structures have been determined for the cytosolic SHMTs of human (24), rabbit (25), and mouse (26) and for E. coli (27) and Bacillus stearothermophilus (28) SHMTs.…”

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confidence: 99%

Location of the Pteroylpolyglutamate-binding Site on Rabbit Cytosolic Serine Hydroxymethyltransferase

Fu¹,

Scarsdale

Kazanina

et al. 2003

Journal of Biological Chemistry

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Serine hydroxymethyltransferase (SHMT; EC 2.1.2.1) catalyzes the reversible interconversion of serine and glycine with transfer of the serine side chain one-carbon group to tetrahydropteroylglutamate (H 4 PteGlu), and also the conversion of 5,10-methenyl-H 4 PteGlu to 5-formyl-H 4 PteGlu. In the cell, H 4 PteGlu carries a poly-␥-glutamyl tail of at least 3 glutamyl residues that is required for physiological activity. This study combines solution binding and mutagenesis studies with crystallographic structure determination to identify the extended binding site for tetrahydropteroylpolyglutamate on rabbit cytosolic SHMT. Equilibrium binding and kinetic measurements of H 4 PteGlu 3 and H 4 PteGlu 5 with wild-type and Lys 3 Gln or Glu site mutant homotetrameric rabbit cytosolic SHMTs identified lysine residues that contribute to the binding of the polyglutamate tail. The crystal structure of the enzyme in complex with 5-formyl-H 4 PteGlu 3 confirms the solution data and indicates that the conformation of the pteridine ring and its interactions with the enzyme differ slightly from those observed in complexes of the monoglutamate cofactor. The polyglutamate chain, which does not contribute to catalysis, exists in multiple conformations in each of the two occupied binding sites and appears to be bound by the electrostatic field created by the cationic residues, with only limited interactions with specific individual residues.

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Interactions of pig liver serine hydroxymethyltransferase with methyl tetrahydropteroylpolyglutamate inhibitors and with tetrahydropteroylpolyglutamate substrates

Cited by 55 publications

References 23 publications

Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

Kinetic Mechanism and the Rate-limiting Step of Plasmodium vivax Serine Hydroxymethyltransferase

Location of the Pteroylpolyglutamate-binding Site on Rabbit Cytosolic Serine Hydroxymethyltransferase

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