Labilization of the Î±-Hydrogen of Amino-acids in the Presence of Aminopherase

Konikova, A. S.; Dobbert, N. N.; Braunstein, A. E.

doi:10.1038/159067a0

Cited by 33 publications

(11 citation statements)

References 11 publications

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“…Recently, an alternative approach was introduced for measuring in vivo synthesis rates of individual proteins termed "proteome dynamics," where the precursor amino acid pool is labeled by administration of heavy water ( 2 H 2 O) (6,10,11,16,21,31). 2 H 2 O generates 2 H-labeled amino acids via transamination and/or de novo synthesis without perturbing the concentration of precursor amino acids (19). This approach labels all newly synthesized proteins and enables measurement on a proteome scale.…”

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confidence: 99%

Assessment of cardiac proteome dynamics with heavy water: slower protein synthesis rates in interfibrillar than subsarcolemmal mitochondria

Kasumov

Dabkowski

Shekar

et al. 2013

American Journal of Physiology-Heart and Circulatory Physiology

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Traditional proteomics provides static assessment of protein content, but not synthetic rates. Recently, proteome dynamics with heavy water ((2)H2O) was introduced, where (2)H labels amino acids that are incorporated into proteins, and the synthesis rate of individual proteins is calculated using mass isotopomer distribution analysis. We refine this approach with a novel algorithm and rigorous selection criteria that improve the accuracy and precision of the calculation of synthesis rates and use it to measure protein kinetics in spatially distinct cardiac mitochondrial subpopulations. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated from adult rats, which were given (2)H2O in the drinking water for up to 60 days. Plasma (2)H2O and myocardial (2)H-enrichment of amino acids were stable throughout the experimental protocol. Multiple tryptic peptides were identified from 28 proteins in both SSM and IFM and showed a time-dependent increase in heavy mass isotopomers that was consistent within a given protein. Mitochondrial protein synthesis was relatively slow (average half-life of 30 days, 2.4% per day). Although the synthesis rates for individual proteins were correlated between IFM and SSM (R(2) = 0.84; P < 0.0001), values in IFM were 15% less than SSM (P < 0.001). In conclusion, administration of (2)H2O results in stable enrichment of the cardiac precursor amino acid pool, with the use of refined analytical and computational methods coupled with cell fractionation one can measure synthesis rates for cardiac proteins in subcellular compartments in vivo, and protein synthesis is slower in mitochondria located among the myofibrils than in the subsarcolemmal region.

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confidence: 99%

Assessment of cardiac proteome dynamics with heavy water: slower protein synthesis rates in interfibrillar than subsarcolemmal mitochondria

Kasumov

Dabkowski

Shekar

et al. 2013

American Journal of Physiology-Heart and Circulatory Physiology

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“…1). The enzymes of intermediary metabolism transfer 2 H from body water to nonessential amino acids, whereas all free amino acids, including essential amino acids, are labeled through transamination (16). Recently we demonstrated that the steady state 2 H labeling of most intracellular amino acids was achieved within 45 min of an intraperitoneal bolus of 2 H 2 O, suggesting that the transfer of amino acids to the polypeptide chain is the rate-limiting step in protein biosynthesis (12,17).…”

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“…Recently we and others developed the 2 H 2 O metabolic labeling technique to measure protein turnover in free living animals (11)(12)(13)(14)(15). In rats that have been given intraperitoneal injection of 2 H 2 O, 2 H equilibrates with total body water and amino acids such as alanine within 10 and 20 min, respectively (16) (Fig. 1).…”

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Plasma Proteome Dynamics: Analysis of Lipoproteins and Acute Phase Response Proteins with 2H2O Metabolic Labeling

Willard

Rachdaoui

et al. 2012

Molecular & Cellular Proteomics

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Understanding the pathologies related to the regulation of protein metabolism requires methods for studying the kinetics of individual proteins. We developed a 2 H 2 O metabolic labeling technique and software for protein kinetic studies in free living organisms. This approach for proteome dynamic studies requires the measurement of total body water enrichments by GC-MS, isotopic distribution of the tryptic peptide by LC-MS/MS, and estimation of the asymptotical number of deuterium incorporated into a peptide by software. We applied this technique to measure the synthesis rates of several plasma lipoproteins and acute phase response proteins in rats. Samples were collected at different time points, and proteins were separated by a gradient gel electrophoresis.2 H labeling of tryptic peptides was analyzed by ion trap tandem mass spectrometry (LTQ MS/MS) for measurement of the fractional synthesis rates of plasma proteins. The high sensitivity of LTQ MS in zoom scan mode in combination with 2 H label amplification in proteolytic peptides allows detection of the changes in plasma protein synthesis related to animal nutritional status. Our results demonstrate that fasting has divergent effects on the rate of synthesis of plasma proteins, increasing synthesis of ApoB 100 but decreasing formation of albumin and fibrinogen. We conclude that this technique can effectively measure the synthesis of plasma proteins and can be used to study the regulation of protein homeostasis under physiological and pathological conditions.

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“…Pyridoxal-P-phosphate (PLP)-dependent lyases displaying broad substrate specificity are able to catalyze stereospecific isotope exchange of a-protons of various amino acids [1][2][3][4] including both real substrates and reversible competitive inhibitors, which do not change their chemical identities under the action of the enzyme. The exchange is usually performed in heavy water, and proceeds with a complete retention of the natural (S)-configuration of amino acids.…”

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The mechanism of α‐proton isotope exchange in amino acids catalysed by tyrosine phenol‐lyase

Faleev

Demidkina

Tsvetikova

et al. 2004

European Journal of Biochemistry

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To shed light on the mechanism of isotopic exchange of a-protons in amino acids catalyzed by pyridoxal phosphate (PLP)-dependent enzymes, we studied the kinetics of quinonoid intermediate formation for the reactions of tyrosine phenol-lyase with L-phenylalanine, L-methionine, and their a-deuterated analogues in D 2 O, and we compared the results with the rates of the isotopic exchange under the same conditions. We have found that, in the L-phenylalanine reaction, the internal return of the a-proton is operative, and allowing for its effect, the exchange rate is accounted for satisfactorily. Surprisingly, for the reaction with L-methionine, the enzymatic isotope exchange went much faster than might be predicted from the kinetic data for quinonoid intermediate formation. This result allows us to suggest the existence of an alternative, possibly concerted, mechanism of a-proton exchange.Keywords: amino acids; isotopic exchange; mechanism; a-proton; tyrosine phenol-lyase.Pyridoxal-P-phosphate (PLP)-dependent lyases displaying broad substrate specificity are able to catalyze stereospecific isotope exchange of a-protons of various amino acids [1-4] including both real substrates and reversible competitive inhibitors, which do not change their chemical identities under the action of the enzyme. The exchange is usually performed in heavy water, and proceeds with a complete retention of the natural (S)-configuration of amino acids. The characteristic PLP-dependent enzymes in this respect are tyrosine phenol-lyase (TPL) (EC 4.1.99.2), tryptophan indole-lyase (EC 4.1.99.1), and L-methionine-c-lyase (EC 4.4.1.11). These enzymes are used as very effective biocatalysts for preparation of enantiomerically pure a-deuterated (S)-amino acids [5][6][7].In the framework of the generally accepted notions of mechanisms of PLP-dependent enzymes the mechanism of the isotopic exchange traditionally is considered to be associated with formation of quinonoid intermediates (Scheme 1). In the holoenzymes (E) the cofactor PLP is bound in the active site as an Ôinternal aldimineÕ with an e-amino group of a definite lysine residue. As a result of interaction with an amino acid substrate, or inhibitor, the internal aldimine (E) is substituted by an ÔexternalÕ one (ES), which undergoes the abstraction of the a-proton by a certain enzyme group, leading to formation of a Ôquinonoid intermediateÕ (EA). The reversibility of the latter transformation should lead in heavy water to the isotopic exchange of the a-proton if the abstracted proton may be easily exchanged with the solvent. However, the kinetics of quinonoid formation was examined until now only in water solutions [8][9][10][11], while measurements in heavy water, in conditions identical to those of the isotopic exchange, were not performed. No attempts to quantitatively estimate the rates of the exchange of the abstracted proton in the active site have been reported. We have noted earlier [8] that no direct correlation was observed between the amount of the quinonoid intermediate formed und...

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Labilization of the Î±-Hydrogen of Amino-acids in the Presence of Aminopherase

Cited by 33 publications

References 11 publications

Assessment of cardiac proteome dynamics with heavy water: slower protein synthesis rates in interfibrillar than subsarcolemmal mitochondria

Assessment of cardiac proteome dynamics with heavy water: slower protein synthesis rates in interfibrillar than subsarcolemmal mitochondria

Plasma Proteome Dynamics: Analysis of Lipoproteins and Acute Phase Response Proteins with 2H2O Metabolic Labeling

The mechanism of α‐proton isotope exchange in amino acids catalysed by tyrosine phenol‐lyase

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