Proton transfer in catalysis by fumarase

Rose, Irwin A.; Warms, Jessie V.B.; Kuo, Donald J.

doi:10.1021/bi00156a019

Cited by 34 publications

(43 citation statements)

References 18 publications

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“…2), the final release of DHAP (step VII) is discussed to be the rate-limiting step of the whole sequence (37,38), however, on the other side in the performed V max /K m,FBP experiments, only the reactions before the first irreversible step, here the release of glyceraldehyde 3-P (step IV), can contribute to the observed isotope effects. Intriguingly we found on C-3 a kinetic isotope effect significantly different from unity, while the one on C-4 was practically unity.…”

Section: Discussionmentioning

confidence: 93%

Carbon Isotope Effects on the Fructose-1,6-bisphosphate Aldolase Reaction, Origin for Non-statistical 13C Distributions in Carbohydrates

Gleixner

Schmidt

1997

Journal of Biological Chemistry

120

107

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The kinetic and equilibrium isotope effects on the fructose-1,6-bisphosphate aldolase reaction have been determined using the rabbit muscle enzyme. The natural 13 C abundance for both atoms participating in the bond splitting were measured in position C-1 of dihydroxyacetone phosphate and glyceraldehyde 3-P after irreversible conversion to glycerol-3-P and 3-phosphoglycerate, respectively, and chemical degradation. The carbon isotope effects were determined comparing the 13 C content of the corresponding positions after partial and complete turnover, and after complete equilibration of the reactants. 13 (V max /K m ) on C-3 was 1.016 ؎ 0.007 and 0.997 ؎ 0.009 on position C-4, and the equilibrium isotope effects K 12 /K 13 on these positions were 1.0036 ؎ 0.0002 and 1.0049 ؎ 0.0001.The observed kinetic isotope effect on C-3 is discussed to originate from the formation of the enamine, which comes to equilibrium before the rate determining release of glyceraldehyde 3-P from the ternary complex. The equilibrium isotope effect is seen as the reason for an earlier-found relative 13 C enrichment in position C-3 and C-4 of glucose and for varying enrichments in 13 C of carbohydrates from different compartments of cells. The kinetic isotope effect is suggested to cause 13 C discriminations in the C-3 pool in context with the hexose formation in competition with other dihydroxyacetone phosphate turnover reactions.The relative enrichment of carbon-13 in the carboxyl group of amino acids, as observed by Abelson and Hoering (1) in 1961, was the first indication for the existence of non-statistical isotope distributions in biological compounds. Later results on acetic acid (2) and on acetoin (3) gave evidence that this observation was just one example of a common phenomenon. In order to find a general explanation for this observation Galimov (4) discussed that even in chemically unequilibrated systems, e.g. biological systems, a microscopic reversibility in enzymatic reactions is the origin for a thermodynamically ordered isotope distribution. The author's calculations could in fact explain some of the isotopic patterns of natural compounds known at that time. However, the presumption of a general thermodynamic equilibrium in biological systems is probably not realistic. In our opinion kinetic isotope effects on enzymatic reactions should be considered as primary causes for isotope discriminations. This has been proven for the primary CO 2 -fixing reactions (5-8). In secondary metabolism, the isotope effect on the pyruvate dehydrogenase reaction has been made responsible for the general depletion of 13 C in metabolites of acetyl-CoA, such as fatty acids or isoprenoids (9 -15).More detailed interpretations were possible when the total isotopic patterns of primary and secondary metabolites became available. Especially, our corresponding investigations on glucose indicated an enrichment of 13 C in positions C-3 and C-4, and a depletion of 13 C in positions C-1 and C-6 (Fig. 1) of this important primary metabolite (16). We have a...

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Section: Discussionmentioning

confidence: 93%

Carbon Isotope Effects on the Fructose-1,6-bisphosphate Aldolase Reaction, Origin for Non-statistical 13C Distributions in Carbohydrates

Gleixner

Schmidt

1997

Journal of Biological Chemistry

120

107

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“…Mitochondrial hexokinase activity is also increased in many tumors (Bustamante and Pedersen, 1977;Parry and Pedersen, 1983) where it has been reported to correlate with in vivo tumor growth rates (Bustamante et al, 1981). The dynamic movement of hexokinase between mitochondrial and cytosolic compartments is influenced by a variety of factors that include physiologic concentrations of glucose-6-phosphate, ATP, divalent cations, inorganic phosphate and intracellular pH (Rose and Warms, 1967;Wilson, 1978;Miccoli et al, 1996Miccoli et al, , 1998. By providing 'privileged access' to intramitochondrial ATP, mitochondrial localization is thought to confer specific functional advantages to mitochondria-bound hexokinases.…”

Section: Mammalian Cells Express Multiple Hexokinase Isoformsmentioning

confidence: 99%

“…The potential ability of other ATP-dependent phosphotransferases to substitute for mitochondrial hexokinases in preventing apoptosis Glucokinase It is widely accepted that GK, which lacks a putative hydrophobic amino-terminal mitochondrial binding domain, is incapable of binding mitochondria in the liver (Rose and Warms, 1967;Wilson and Felgner, 1977;Ureta et al, 1983;Arora and Pedersen, 1988;MalaisseLagae and Malaisse, 1988;Shinohara et al, 1997;Wilson, 2003;Bustamante et al, 2005). However, a recent report of direct interaction between the proapoptotic 'BH3-only' protein Bad and GK in liver cells has been interpreted as suggestive of interplay between apoptotic proteins and glucose metabolism in the liver reminiscent of that described for mitochondrial hexokinases in other cell types (Danial et al, 2003).…”

Section: Gsh Peroxide Inactivationmentioning

confidence: 99%

Mitochondrial hexokinases, novel mediators of the antiapoptotic effects of growth factors and Akt

2006

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Cell survival has been closely linked to both trophic growth factor signaling and cellular metabolism. Such couplings have obvious physiologic and pathophysiologic implications, but their underlying molecular bases remain incompletely defined. As a common mediator of both the metabolic and anti-apoptotic effects of growth factors, the serine/threonine kinase Akt -also known as protein kinase B or PKB -is capable of regulating and coordinating these inter-related processes. The glucose dependence of the antiapoptotic effects of growth factors and Akt plus a strong correlation between Akt-regulated mitochondrial hexokinase association and apoptotic susceptibility suggest a major role for hexokinases in these effects. Mitochondrial hexokinases catalyse the first obligatory step of glucose metabolism and directly couple extramitochondrial glycolysis to intramitochondrial oxidative phosphorylation, and are thus well suited to play this role. The ability of Akt to regulate energy metabolism appears to have evolutionarily preceded the capacity to control cell survival. This suggests that Akt-dependent metabolic regulatory functions may have given rise to glucose-dependent antiapoptotic effects that evolved as an adaptive sensing system involving hexokinases and serve to ensure mitochondrial homeostasis, thereby coupling metabolism to cell survival. We hypothesize that the enlistment of Akt and hexokinase in the control of mammalian cell apoptosis evolved as a response to the recruitment of mitochondria to the apoptotic cascade. The central importance of mitochondrial hexokinases in cell survival also suggests that they may represent viable therapeutic targets in cancer.

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“…The enzyme must stabilize the enamine 2 and/or the preceding iminium 1 such that no decomposition occurs prior to reaction with the aldehyde as shown in 3. This stability is reflected in solution where the enzymatic populations 1 and 2 represent 20 and 60%, respectively, of bound DHAP on the muscle enzyme under equilibrium conditions (9). The interconversion between the two forms implicates the conserved C-terminal Tyr 363 residue whose proteolysis inhibits the stereospecific proton exchange step, making it rate-limiting (10), whereas the penultimate residues (357-362) of the C-terminal region modulate the rate of exchange reaction (11).…”

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confidence: 99%

“…To investigate the mechanism of stereoselective proton transfer in class I aldolases and how the enzyme is able to make efficient use of intermediates that are intrinsically unstable molecules, high resolution crystallographic studies were undertaken of rabbit muscle class I FBP aldolase in complex with DHAP using aldolase crystals incubated in the presence of DHAP in non-acidic buffer (pH 7.5), similar to kinetic studies (9). Flash freezing of a native rabbit muscle aldolase crystal soaked in a saturating DHAP solution trapped the enamine intermediate.…”

mentioning

confidence: 99%

Stereospecific Proton Transfer by a Mobile Catalyst in Mammalian Fructose-1,6-bisphosphate Aldolase

St-Jean¹,

Sygusch

2007

Journal of Biological Chemistry

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Class I fructose-1,6-bisphosphate aldolases catalyze the interconversion between the enamine and iminium covalent enzymatic intermediates by stereospecific exchange of the pro(S) proton of the dihydroxyacetone-phosphate C3 carbon, an obligatory reaction step during substrate cleavage. To investigate the mechanism of stereospecific proton exchange, high resolution crystal structures of native and a mutant Lys 146 Stereospecificity is one of the hallmarks of enzyme catalysis. Aldolases, which are abundant and ubiquitous enzymes, catalyze stereospecific carbon-carbon bond formation, one of the most important transformations in living organisms. Their role is best known in glycolysis where fructose-1,6-bis(phosphate) (FBP) 3 aldolases (EC 4.1.2.13) promote the cleavage of FBP to triose phosphates, D-glyceraldehyde-3-phosphate (G3P), and dihydroxyacetone phosphate (DHAP). A common feature to class I enzymes is the use of a covalent mechanism for catalysis implicating iminium (protonated Schiff base) formation between a lysine residue on the enzyme and a ketose substrate (1) that entails stereospecific proton exchange in the covalent intermediate (2). Of the three aldolase isozymes found in vertebrates (3), the catalytic mechanism has been extensively studied using class I aldolase A from rabbit muscle and key intermediates are depicted in reaction Scheme 1.In the condensation direction, the reaction involves covalent intermediate formation with the keto triose phosphate DHAP followed by condensation with the aldehyde G3P to form the ketose of the acyclic FBP substrate (4, 5). To form the C3-C4 bond of FBP, the enzyme stereospecifically abstracts the pro(S) C3 proton of the trigonal iminium 1 (6, 7) that is formed from the Michaelis complex with DHAP thereby generating via the enamine 2 (2) the carbanionic character at C3 of DHAP for the aldol reaction. The nascent carbon-carbon bond has the same orientation as the pro(S) ␣-hydrogen initially abstracted from the DHAP imine intermediate. The overall retention of configuration at C3 requires that proton abstraction from 1 to yield the enamine 2 and condensation with aldehyde in 3 must take place from the same direction on the enzyme (8). The iminium intermediate formed is then hydrolyzed and FBP is released by the inverse reaction sequence shown in Scheme 1.A distinguishing mechanistic feature of class I aldolases is the relative stability of the iminium 1 and enamine 2 forms, which is a consequence of the catalytic requirements. The enzyme must stabilize the enamine 2 and/or the preceding iminium 1 such that no decomposition occurs prior to reaction with the aldehyde as shown in 3. This stability is reflected in solution where the enzymatic populations 1 and 2 represent 20 and 60%, respectively, of bound DHAP on the muscle enzyme under equilibrium conditions (9). The interconversion between the two forms implicates the conserved C-terminal Tyr 363 residue whose proteolysis inhibits the stereospecific proton exchange step, making it rate-limiting (10), whereas the pen...

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Proton transfer in catalysis by fumarase

Cited by 34 publications

References 18 publications

Carbon Isotope Effects on the Fructose-1,6-bisphosphate Aldolase Reaction, Origin for Non-statistical 13C Distributions in Carbohydrates

Carbon Isotope Effects on the Fructose-1,6-bisphosphate Aldolase Reaction, Origin for Non-statistical 13C Distributions in Carbohydrates

Mitochondrial hexokinases, novel mediators of the antiapoptotic effects of growth factors and Akt

Stereospecific Proton Transfer by a Mobile Catalyst in Mammalian Fructose-1,6-bisphosphate Aldolase

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