Inverse Solvent Isotope Effects in the NAD-Malic Enzyme Reaction Are the Result of the Viscosity Difference between D2O and H2O: Implications for Solvent Isotope Effect Studies

Karsten, William E.; Lai, Chung-Jeng; Cook, Paul F.

doi:10.1021/ja00127a002

Cited by 78 publications

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“…(32, 33) Furthermore, the lack of a viscosity effect indicates FIH does not undergo a viscosity-sensitive conformational change, which has been shown to obscure SIEs for some enzymes. (34, 35)…”

Section: Resultsmentioning

confidence: 99%

Inverse Solvent Isotope Effects Arising from Substrate Triggering in the Factor Inhibiting Hypoxia Inducible Factor

Saban

Knapp

2013

Biochemistry

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Oxygen homeostasis plays a critical role in angiogenesis, erythropoiesis and cell metabolism. Oxygen homeostasis is set by the hypoxia inducible factor-1α (HIF-1α) pathway, which is controlled by Factor Inhibiting HIF-1α (FIH). FIH is a non-heme Fe(II), α-ketoglutarate dependent dioxygenase that inhibits HIF-1α by hydroxylating the C-terminal transactivation domain (CTAD) of HIF-1α at HIF-Asn803. A tight coupling between CTAD binding and O2-activation is essential for hypoxia sensing, making changes in the coordination geometry of Fe(II) upon CTAD encounter a crucial feature of this enzyme. Although the consensus chemical mechanism for FIH proposes that CTAD binding triggers O2-activation by causing the Fe(II) cofactor to release an aquo ligand, experimental evidence of this point has been absent. More broadly, this proposed coordination change at Fe(II) has not been observed during steady-state turnover in any αKG oxygenase to date. In this manuscript, solvent isotope effects (SIEs) were used as a direct mechanistic probe of substrate triggered aquo release in FIH, as inverse SIEs (SIE < 1) are signatures for pre-equilibrium aquo release from metal ions. Our mechanistic studies of FIH have revealed inverse solvent isotope effects in the steady-state rate constants at limiting concentrations of CTAD or αKG: D2Okcat/KM(CTAD) = 0.40 ± 0.07, D2Okcat/KM(αKG) = 0.32 ± 0.08, providing direct evidence for aquo release during steady-state turnover. Furthermore, the SIE at saturating concentrations of CTAD and αKG was inverse, D2Okcat = 0.51 ± 0.07, indicating that aquo release occurs after CTAD binds. The inverse kinetic SIEs observed in the steady state for FIH can be explained by a strong Fe-OH2 bond. The stable Fe-OH2 bond plays an important part in FIH’s regulatory role over O2 homeostasis in humans, and points toward a strategy for tightly coupling O2-activation with CTAD hydroxylation that relies on substrate triggering.

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Section: Resultsmentioning

confidence: 99%

Inverse Solvent Isotope Effects Arising from Substrate Triggering in the Factor Inhibiting Hypoxia Inducible Factor

Saban

Knapp

2013

Biochemistry

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“…An inverse isotope effect of 0.67 on k cat /K m in D 2 O revealed that reaction in heavy water is faster than that in H 2 O. Inverse SIEs in enzyme-catalyzed reactions are frequently attributed to an acid dissociation constant of a thiol group, dissociation of a metal-chelated water molecule, or torsional restrictions (43,44). These possibilities were further analyzed, and the last was found likely for this system because there are no active site cysteines, and the interactions of Mg 2ϩ with solvent are not sufficiently strong to affect fractionation factors of solvating waters (33).…”

Section: Discussionmentioning

confidence: 99%

Dissection of the Stepwise Mechanism to β-Lactam Formation and Elucidation of a Rate-determining Conformational Change in β-Lactam Synthetase

Raber

Freeman

Townsend

2009

Journal of Biological Chemistry

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Clavulanic acid is a widely used ␤-lactamase inhibitor whose key ␤-lactam core is formed by ␤-lactam synthetase. ␤-Lactam synthetase exhibits a Bi-Ter mechanism consisting of two chemical steps, acyl-adenylation followed by ␤-lactam formation. 32 PP i -ATP exchange assays showed the first irreversible step of catalysis is acyl-adenylation. From a small, normal solvent isotope effect (1.38 ؎ 0.04), it was concluded that ␤-lactam synthesis contributes at least partially to k cat . Site-specific mutation of Lys-443 identified this residue as the ionizable group at pK a ϳ 8.1 apparent in the pH-k cat profile that stabilizes the ␤-lactamforming step. Viscosity studies demonstrated that a protein conformational change was also partially rate-limiting on k cat attenuating the observed solvent isotope effect on ␤-lactam formation. Adherence to Kramers' theory gave a slope of 1.66 ؎ 0.08 from a plot of log( o k cat /k cat ) versus log(/ o ) consistent with opening of a structured loop visible in x-ray data preceding product release. Internal "friction" within the enzyme contributes to a slope of >1 in this analysis. Correspondingly, earlier in the catalytic cycle ordering of a mobile active site loop upon substrate binding was manifested by an inverse solvent isotope effect (0.67 ؎ 0.15) on k cat /K m . The increased second-order rate constant in heavy water was expected from ordering of this loop over the active site imposing torsional strain. Finally, an Eyring plot displayed a large enthalpic change accompanying loop movement (⌬H ‡ ϳ 20 kcal/mol) comparable to the chemical barrier of ␤-lactam formation.Resistance to antibiotics is a consequence of their widespread use in the treatment of disease and can arise with dispiriting rapidity after the introduction of a new drug. ␤-Lactam antibiotics constitute 55% of all antibiotics sold despite their prominent use for more than 50 years (1). Their longevity is because of semi-synthetic structural modifications, the discovery of new members of the family from natural sources, and the growing importance of inhibitors of the principal means of resistance, the ␤-lactamases (2). Clavulanic acid is a potent serine ␤-lactamase inhibitor isolated from Streptomyces clavuligerus (3) whose combination with broad spectrum ␤-lactam antibiotics has proven to be highly effective against resistant infections in humans (4, 5). Inhibitor-resistant ␤-lactamases are emerging (1), however, and the need for new entities clinically useful against them is growing.Clavulanic acid is one of only three ␤-lactamase inhibitors in clinical use (Scheme 1) (6). Although other clavams produced in nature may possess antibacterial and antifungal properties, clavulanic acid is the only known clavam with potent ␤-lactamase inhibitory activity owing in part to its 3R,5R stereochemistry (7). It is therefore important to understand the steps involved in this pathway to enable the production of derivatives. Substitution at C-6 of clavulanic acid derivatives, for example, led to broader spectrum inhibition toward ␤...

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“…24 for more examples). Such inverse SIEs have been interpreted as being the result of the increased viscosity of D 2 O solutions (26), the dissociation of an exchangeable hydrogen from a thiol group, which has a fractionation factor of less than 1 (27), medium effects where SIEs result from changes in solvation of solutes in D 2 O versus H 2 O (28), the dissociation of a metalchelated water molecule (27), or the result of restrictions on the torsional movement of the affected bond (29). Although the Asn 195 3 Ala mutant did show an inverse SVE, the levels at the concentration of glucose matching the viscosity of D 2 O (ϳ9%) were not sufficient to match the observed inverse SIE.…”

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confidence: 99%

Molecular Mechanism of Aminoglycoside Antibiotic Kinase APH(3′)-IIIa

Boehr¹,

Thompson²,

Wright

2001

Journal of Biological Chemistry

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The aminoglycoside antibiotic kinases (APHs) constitute a clinically important group of antibiotic resistance enzymes. APHs share structural and functional homology with Ser/Thr and Tyr kinases, yet only five amino acids are invariant between the two groups of enzymes and these residues are all located within the nucleotide binding regions of the proteins. We have performed sitedirected mutagenesis on all five conserved residues in the aminoglycoside kinase APH (3)

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Inverse Solvent Isotope Effects in the NAD-Malic Enzyme Reaction Are the Result of the Viscosity Difference between D2O and H2O: Implications for Solvent Isotope Effect Studies

Cited by 78 publications

References 0 publications

Inverse Solvent Isotope Effects Arising from Substrate Triggering in the Factor Inhibiting Hypoxia Inducible Factor

Inverse Solvent Isotope Effects Arising from Substrate Triggering in the Factor Inhibiting Hypoxia Inducible Factor

Dissection of the Stepwise Mechanism to β-Lactam Formation and Elucidation of a Rate-determining Conformational Change in β-Lactam Synthetase

Molecular Mechanism of Aminoglycoside Antibiotic Kinase APH(3′)-IIIa

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