A Very Short Hydrogen Bond Provides Only Moderate Stabilization of an Enzyme-Inhibitor Complex of Citrate Synthase

Usher, K.C.; Remington, S.J.; Martin, David P.; Drueckhammer, Dale G.

doi:10.1021/bi00191a002

Cited by 96 publications

(131 citation statements)

References 31 publications

Supporting

Mentioning

116

Contrasting

Unclassified

Order By: Relevance

“…The acetyl-CoA enolate is structurally analogous to CMCoA or CMX (Figure 2), which form isoelectronic ternary complexes because tight binding of anionic-terminus analogues requires uptake of a single proton from solution (19, 27). Crystallographic and solid-state NMR studies show that CMX forms a short hydrogen bond with Asp317 (21, 27, 28) and that the shared proton in the short hydrogen bond resides mainly on the side chain of this active site base (27). A similar configuration is expected in the enolate intermediate: the proton resides mainly on the active site base while the charge resides mainly on the acetyl-CoA enolate (15).…”

Section: Discussionmentioning

confidence: 99%

The Partial Substrate Dethiaacetyl-Coenzyme A Mimics All Critical Carbon Acid Reactions in the Condensation Half-Reaction Catalyzed by Thermoplasma acidophilum Citrate Synthase

et al. 2009

View full text Add to dashboard Cite

Citrate synthase (CS) performs two half-reactions: the mechanistically intriguing condensation of acetyl-CoA with oxaloacetate (OAA) to form citryl-CoA, and the subsequent, slower hydrolysis of citryl-CoA that generally dominates steady-state kinetics. The condensation reaction requires the abstraction of a proton from the methyl carbon of acetyl-CoA to generate a reactive enolate intermediate. The carbanion of that intermediate then attacks the OAA carbonyl to furnish citryl-CoA, the initial product. Using stopped-flow and steady-state fluorescence methods, kinetic substrate isotope effects, and mutagenesis of active site residues, we show that all of the processes that occur in the condensation half-reaction performed by Thermoplasma acidophilum citrate synthase (TpCS) with the natural thioester substrate, acetyl-CoA, also occur with the ketone inhibitor dethiaacetyl-CoA. Free energy profiles demonstrate that the non-hydrolysable product of the condensation reaction, dethiacitryl-CoA, forms a particularly stable complex with TpCS but not pig heart CS.

show abstract

Section: Discussionmentioning

confidence: 99%

The Partial Substrate Dethiaacetyl-Coenzyme A Mimics All Critical Carbon Acid Reactions in the Condensation Half-Reaction Catalyzed by Thermoplasma acidophilum Citrate Synthase

et al. 2009

View full text Add to dashboard Cite

show abstract

“…In the enolized intermediate the close pK match between the enol and His-274 should permit a LBHB to form, thus producing the energy for enolization (1,3). An x-ray study of citrate synthase with bound carboxyl or amide analogs of acetyl-CoA showed a very short (2.4 -2.5 Å) hydrogen bond between the carboxyl or amide group (which corresponds to the methyl carbon of acetyl-CoA) and Asp-375 (45). Although the dissociation constant of the amide was pH-independent, that of the carboxyl inhibitor decreased as the pH was lowered, showing that the carboxyl must be protonated, with the proton presumably between Asp-375 and the carboxyl of the inhibitor.…”

Section: Minireview: Low Barrier Hydrogen Bond In Enzymatic Catalysismentioning

confidence: 99%

The Low Barrier Hydrogen Bond in Enzymatic Catalysis

Cleland¹,

Frey²,

Gerlt³

1998

Journal of Biological Chemistry

533

499

View full text Add to dashboard Cite

The proposal that low barrier (i.e. short, very strong) hydrogen bonds (LBHBs) 1 play a role in enzymatic catalysis was first put forth in 1993 and 1994 (1-4). The proposal was accepted by some but rejected by others (5-8). Initial rejection on theoretical grounds has been followed by increasing experimental support, and recent improvements in theory have been able to account for the experimental observations of LBHBs in enzymes (9 -15). In this minireview we will explain the original proposal, summarize the experimental data from the past few years, and argue that LBHBs do play important roles in enzymatic reactions. Properties of Hydrogen BondsThe strength of a hydrogen bond depends on its length and linearity, the nature of its microenvironment, and the degree to which the pK values of the conjugate acids of the heavy atoms sharing the proton are matched. In water, the hydrogen-bonded oxygens are separated by ϳ2.8 Å, and the ⌬H of formation is ϳ5 kcal mol Ϫ1 . The hydrogen bonds in water are, however, weak because of the poor pK match between the participating oxygen atoms. Because the pKs of H 3 O ϩ and H 2 O are Ϫ1.7 and 15.7, respectively, the proton in the structure H 2 O⅐⅐⅐H-OH is tightly associated with the OH Ϫ group as a water molecule. In the gas phase, where the dielectric constant is low, hydrogen bonds between heteroatoms with matched pKs can be very short and strong, and experimental as well as calculated values of ⌬H of formation can approach 25 or 30 kcal mol Ϫ1 (16, 17). Likewise, in crystals hydrogen bonds can be very strong. The O-O distance in the ion [H- O⅐⅐⅐H⅐⅐⅐O-H] -in a crystal of a chromium complex is only 2.29 Å (18,19). In organic solvents, strong hydrogen bonds can also form, although the ⌬H of formation probably never exceeds 20 kcal mol Ϫ1. Recent calculations suggest that once the dielectric constant is at least 6 the strength of a strong hydrogen bond levels off at a level about half that in the gas phase (13,17). Because the active site of an enzyme is no longer aqueous once it has closed around a substrate, the properties of hydrogen bonds in organic solvents are highly pertinent to enzymatic catalysis.What happens energetically as hydrogen bonds become shortened can be seen in Fig. 1. Structure A represents the situation in water, where the hydrogen is firmly attached to either the lefthand or right-hand oxygen and is more loosely bonded to the other one, with an O-O distance of Ն2.8 Å. There is an energy barrier between the two possible positions of the hydrogen, with the zero point energy levels shown in Fig. 1. Such a hydrogen bond is essentially electrostatic, and the covalent O-H bond is the usual 0.9 -1.0 Å in length.As the overall O-O distance is shortened, the energy barrier drops until it reaches the zero point energy level at an O-O distance of ϳ2.5 Å (Fig. 1B); this is a LBHB. The ⌬H of formation has increased to 15-20 kcal/mol, and the hydrogen can now move freely between the two oxygens. In crystals containing LBHBs, neutron diffraction shows the hydrogen dif...

show abstract

“…Experimental results show that while unusually short hydrogen bonds can be formed in enzymes, including citrate synthase, they do not lead to exceptionally large binding energies [76,77]. The strongest, shortest hydrogen bonds are found for charged interactions between groups with approximately equal pK a 's [51,78], but there is no special stabilization associated with disappearance of the barrier to proton transfer [42], or at DpK a 0 [79].…”

Section: Discussionmentioning

confidence: 98%

Ab initio QM/MM modelling of acetyl-CoA deprotonation in the enzyme citrate synthase

Kamp

Perruccio

Mulholland

2007

Journal of Molecular Graphics and Modelling

View full text Add to dashboard Cite

A Very Short Hydrogen Bond Provides Only Moderate Stabilization of an Enzyme-Inhibitor Complex of Citrate Synthase

Cited by 96 publications

References 31 publications

The Partial Substrate Dethiaacetyl-Coenzyme A Mimics All Critical Carbon Acid Reactions in the Condensation Half-Reaction Catalyzed by Thermoplasma acidophilum Citrate Synthase

The Partial Substrate Dethiaacetyl-Coenzyme A Mimics All Critical Carbon Acid Reactions in the Condensation Half-Reaction Catalyzed by Thermoplasma acidophilum Citrate Synthase

The Low Barrier Hydrogen Bond in Enzymatic Catalysis

Ab initio QM/MM modelling of acetyl-CoA deprotonation in the enzyme citrate synthase

Contact Info

Product

Resources

About