The effects of pH on the rates of isotope exchange catalyzed by alanine aminotransferase

Golichowski, Alan M.; Harruff, Richard C.; Jenkins, W. Terry

doi:10.1016/0003-9861(77)90216-8

Cited by 28 publications

(24 citation statements)

References 41 publications

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“…Production of alanine and glutamate was quite sensitive to the presence of deuterium in upstream glucose. We also observed proton exchange in the methyl group of lactate and alanine, previously described in isolated enzyme preparations [18] and now demonstrated in mammalian tissue.…”

Section: Introductionsupporting

confidence: 68%

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The rate of lactate production from glucose in hearts is not altered by per-deuteration of glucose

Funk

Anderson

Wen

et al. 2017

Journal of Magnetic Resonance

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This study was designed to determine whether perdeuterated glucose experiences a kinetic isotope effect (KIE) as glucose passes through glycolysis and is further oxidized in the tricarboxylic acid (TCA) cycle. Metabolism of deuterated glucose was investigated in two groups of perfused rat hearts. The control group was supplied with a 1:1 mixture of [U-13C6]glucose and [1,6-13C2]glucose, while the experimental group received [U-13C6,U-2H7]glucose and [1,6-13C2]glucose. Tissue extracts were analyzed by 1H, 2H and proton-decoupled 13C NMR spectroscopy. Extensive 2H-13C scalar coupling plus chemical shift isotope effects were observed in the proton-decoupled 13C NMR spectra of lactate, alanine and glutamate. A small but measureable (~8%) difference in the rate of conversion of [U-13C6]glucose vs. [1,6-13C2]glucose to lactate, likely reflecting rates of C–C bond breakage in the aldolase reaction, but conversion of [U-13C6]glucose versus [U-13C6,U-2H7]glucose to lactate did not differ. This shows that the presence of deuterium in glucose does not alter glycolytic flux. However, there were two distinct effects of deuteration on metabolism of glucose to alanine and oxidation of glucose in the TCA. First, alanine undergoes extensive exchange of methyl deuterons with solvent protons in the alanine amino transferase reaction. Second, there is a substantial kinetic isotope effect in metabolism of [U-13C6,U-2H7]glucose to alanine and glutamate. In the presence of [U-13C6,U-2H7]glucose, alanine and lactate are not in rapid exchange with the same pool of pyruvate. These studies indicate that the appearance of hyperpolarized 13C-lactate from hyperpolarized [U-13C6,U-2H7]glucose is not substantially influenced by a deuterium kinetic isotope effect.

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Section: Introductionsupporting

confidence: 68%

“…We assumed that once 2 H was released into intracellular water, it was no longer involved in metabolism. 13 C NMR spectra of the extracts were acquired as described previously [18,22–25]. The multiplets in the 13 C NMR spectrum were fit using a mixed Gaussian/Lorentzian lineshape in the TOPSPIN 3.5 Software (Bruker, Germany).…”

Section: Methodsmentioning

confidence: 99%

The rate of lactate production from glucose in hearts is not altered by per-deuteration of glucose

Funk

Anderson

Wen

et al. 2017

Journal of Magnetic Resonance

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“…(ii) The deuteration occurred without any oxo acid additions, contrary to previously published procedures where oxo acid was always present (Cooper, 1976;Babu & Johnston, 1976;Golichowski et al, 1977). Thus the deuteration can occur without any overall transamination and its increase after addition of an oxo acceptor is less than 2-fold.…”

Section: Discussionmentioning

confidence: 66%

“…A major feature of the E. coli aspartate aminotransferase when compared with the mammalian transaminases (Walter et al, 1975;Cooper, 1976;Babu & Johnston, 1976;Golichowski et al, 1977, and literature cited herein) is its broader specificity, extending to various aromatic amino acids (Chesne & Pelmont, 1973). This is illustrated by the spectral shift of the light-absorption of the enzyme with either L-aspartate or L-phenylalanine (Fig.…”

Section: Discussionmentioning

confidence: 86%

“…spectroscopy (Oshima & Tamiya, 1961), mass spectroscopy (Hilton et al, 1956), 3H exchange (Walter et al, 1975) or p.m.r. spectroscopy (Walter et al, 1975;Whelan & Long, 1969;Cooper, 1976;Babu & Johnston, 1976;Golichowski et al, 1977) when different mammalian amino acid-oxo acid aminotransferases were studied in deuterated water. Enzyme-catalysed exchange of the fl-hydrogen atoms has been reported only for alanine of the amino acids (Walter et al, 1975;Cooper, 1976;Babu & Johnston, 1976; Golichowski et al, 1977), and for oxaloacetate of the oxo acids by Whelan & Long (1969).…”

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confidence: 99%

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Kinetic studies with the use of proton-magnetic-resonance spectroscopy of the specific α-deuteration of amino acids by Escherichia coli aspartate aminotransferase

Gout¹,

Chesne²,

Béguin³

et al. 1978

Biochemical Journal

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Escherichia coli aspartate aminotransferase was exposed to aspartate or phenylalanine without oxo acid in buffered 2H2O. The alpha-hydrogen of the amino acids underwent first-order exchange with respect to both substrate and enzyme. P.m.r. spectroscopy gave consistent reaction-rate constants. The deuterium-exchange rate was only moderately increased by addition of oxo acids and was of the same order as the transamination rate. No beta-deuteration was observed. The C(alpha)-H-bond-breaking step is discussed as a part of the entire transamination mechanism.

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Putrescine metabolism: Enzymatic formation and non-enzymatic isotope exchange of Δ′-pyrroline

Callery

Nayar

Geelhaar

1984

Biol. Mass Spectrom.

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The deamination of putrescine catalysed by diamine oxidase was carried out in deuterium oxide and deuterated buffers. Enamine and alpha, beta-unsaturated intermediates were excluded, based on the observation that deuterium was not incorporated into delta 1-pyrroline during its enzymatic formation in deuterium oxide. When the reaction mixture was buffered with phosphate, isolated delta 1-pyrroline contained two deuterium atoms at C-3, indicating that a phosphate-promoted, non-enzymatic isotope exchange had occurred. Using 5,5-dimethyl-delta 1-pyrroline as a model compound, the nature of the non-enzymatic deuterium exchange was studied and a bifunctional catalysis mechanism proposed. The results suggest that the choice of buffer could alter the conclusions drawn from enzyme mechanism studies involving imine-enamine tautomerism .

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The effects of pH on the rates of isotope exchange catalyzed by alanine aminotransferase

Cited by 28 publications

References 41 publications

The rate of lactate production from glucose in hearts is not altered by per-deuteration of glucose

The rate of lactate production from glucose in hearts is not altered by per-deuteration of glucose

Kinetic studies with the use of proton-magnetic-resonance spectroscopy of the specific α-deuteration of amino acids by Escherichia coli aspartate aminotransferase

Putrescine metabolism: Enzymatic formation and non-enzymatic isotope exchange of Δ′-pyrroline

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