Tina L. Amyes

Proton transfer at carbon.

The viability of living systems requires that C--H bonds of biological molecules be stable in water, but that there also be a mechanism for shortening the timescale for their heterolytic cleavage through enzymatic catalysis of a variety of catabolic and metabolic reactions. An understanding of the mechanism of enzymatic catalysis of proton transfer at carbon requires the integration of results of studies to determine the structure of the enzyme-substrate complex with model studies on the mechanism for the non-enzymatic reaction in water, and the effect of the local protein environment on the stability of the transition state for this reaction. A common theme is the importance of electrostatic interactions in providing stabilization of bound carbanion intermediates of enzyme-catalyzed proton-transfer reactions.

A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD+) by Phosphite Dianion†

The ratio of the second-order rate constants for reduction of dihydroxyacetone phosphate (DHAP) and of the neutral truncated substrate glycolaldehyde (GLY) by glycerol 3-phosphate dehydrogenase (NAD (+), GPDH) saturated with NADH is (1.0 x 10 (6) M (-1) s (-1))/(8.7 x 10 (-3) M (-1) s (-1)) = 1.1 x 10 (8), which was used to calculate an intrinsic phosphate binding energy of at least 11.0 kcal/mol. Phosphite dianion binds very weakly to GPDH ( K d > 0.1 M), but the bound dianion strongly activates GLY toward enzyme-catalyzed reduction by NADH. Thus, the large intrinsic phosphite binding energy is expressed only at the transition state for the GPDH-catalyzed reaction. The ratio of rate constants for the phosphite-activated and the unactivated GPDH-catalyzed reduction of GLY by NADH is (4300 M (-2) s (-1))/(8.7 x 10 (-3) M (-1) s (-1)) = 5 x 10 (5) M (-1), which was used to calculate an intrinsic phosphite binding energy of -7.7 kcal/mol for the association of phosphite dianion with the transition state complex for the GPDH-catalyzed reduction of GLY. Phosphite dianion has now been shown to activate bound substrates for enzyme-catalyzed proton transfer, decarboxylation, hydride transfer, and phosphoryl transfer reactions. Structural data provide strong evidence that enzymic activation by the binding of phosphite dianion occurs at a modular active site featuring (1) a binding pocket complementary to the reactive substrate fragment which contains all the active site residues needed to catalyze the reaction of the substrate piece or of the whole substrate and (2) a phosphate/phosphite dianion binding pocket that is completed by the movement of flexible protein loop(s) to surround the nonreacting oxydianion. We propose that loop motion and associated protein conformational changes that accompany the binding of phosphite dianion and/or phosphodianion substrates lead to encapsulation of the substrate and/or its pieces in the protein interior, and to placement of the active site residues in positions where they provide optimal stabilization of the transition state for the catalyzed reaction.

Hydron Transfer Catalyzed by Triosephosphate Isomerase. Products of the Direct and Phosphite-Activated Isomerization of [1-13C]-Glycolaldehyde in D2O

Product distributions for the reaction of glycolaldehyde labeled with carbon-13 at the carbonyl carbon ([1-(13)C]-GA) catalyzed by triosephosphate isomerase (TIM) in D(2)O at pD 7.0 in the presence of phosphite dianion and in its absence were determined by (1)H NMR spectroscopy. We observe three products for the relatively fast phosphite-activated reaction (Amyes, T. L., and Richard, J. P. (2007) Biochemistry 46, 5841-5854): [2-(13)C]-GA from isomerization with intramolecular transfer of hydrogen (12% of products), [2-(13)C,2-(2)H]-GA from isomerization with incorporation of deuterium from D(2)O at C-2 (64% of products), and [1-(13)C,2-(2)H]-GA from incorporation of deuterium from D(2)O at C-2 (23% of products). The much slower unactivated reaction in the absence of phosphite results in formation of the same three products along with the doubly deuterated product [1-(13)C,2,2-(2)H(2)]-GA. The two isomerization products ([2-(13)C]-GA and [2-(13)C,2-(2)H]-GA) are formed in the same relative yields in both the unactivated and the phosphite-activated reactions. However, the additional [1-(13)C,2-(2)H]-GA and the doubly deuterated [1-(13)C,2,2-(2)H(2)]-GA formed in the unactivated TIM-catalyzed reaction are proposed to result from nonspecific reaction(s) at the protein surface. The data provide evidence that phosphite dianion affects the rate, but not the product distribution, of the TIM-catalyzed reaction of [1-(13)C]-GA at the enzyme active site. They are consistent with the conclusion that both reactions occur at an unstable loop-closed form of TIM and that activation of the isomerization reaction by phosphite dianion results from utilization of the intrinsic binding energy of phosphite dianion to stabilize the active loop-closed enzyme.

Concurrent stepwise and concerted substitution reactions of 4-methoxybenzyl derivatives and the lifetime of the 4-methoxybenzyl carbocation

The rates of reaction of 4-methoxybenzyl chloride (1), pentafluorobenzoate, and 3,5-dinitrobenzoate in 50:50 (v/v) trifluoroethanol/water are zero order in the concentration of azide ion. These reactions give good yields of the azide adduct from trapping of the 4-methoxybenzyl carbocation intermediate, and there is also strong common chloride ion inhibition of the reaction of 4-MeOArCH 2 Cl. The yields of the azide and solvent adducts give the rate constant ratio k az /k s =25 M −1 , and analysis of the chloride common ion effect gives k Cl /k s =9 M −1 , for partitioning of the carbocation intermediate. The reaction of azide ion with the carbocation intermediate serves as a diffusion-controlled «clock» for the reactions of solvent and chloride ion. There is a solvent-induced change in mechanism and in the less ionizing solvents of 80:20 (v/v) and 70:30 (v/v) acetone/water; the reaction of azide ion with 1 produces the azide adduct by both trapping of the carbocation intermediate and by a concerted bimolecular substitution reaction

Mechanism for Activation of Triosephosphate Isomerase by Phosphite Dianion: The Role of a Ligand-Driven Conformational Change

The L232A mutation in triosephosphate isomerase (TIM) from Trypanosoma brucei brucei results in a small 6-fold decrease in k(cat)/K(m) for the reversible enzyme-catalyzed isomerization of glyceraldehyde 3-phosphate to give dihydroxyacetone phosphate. In contrast, this mutation leads to a 17-fold increase in the second-order rate constant for the TIM-catalyzed proton transfer reaction of the truncated substrate piece [1-(13)C]glycolaldehyde ([1-(13)C]-GA) in D(2)O, a 25-fold increase in the third-order rate constant for the reaction of the substrate pieces GA and phosphite dianion (HPO(3)(2-)), and a 16-fold decrease in K(d) for binding of HPO(3)(2-) to the free enzyme. Most significantly, the mutation also results in an 11-fold decrease in the extent of activation of the enzyme toward turnover of GA by bound HPO(3)(2-). The data provide striking evidence that the L232A mutation leads to a ca. 1.7 kcal/mol stabilization of a catalytically active loop-closed form of TIM (E(c)) relative to an inactive open form (E(o)). We propose that this is due to the relief, in L232A mutant TIM, of unfavorable steric interactions between the bulky hydrophobic side chain of Leu-232 and the basic carboxylate side chain of Glu-167, the catalytic base, which destabilize E(c) relative to E(o).

Magnitude and origin of the enhanced basicity of the catalytic glutamate of triosephosphate isomerase.

Glu-167 of triosephosphate isomerase from Trypanosoma brucei brucei (TbbTIM) acts as the base to deprotonate substrate to form an enediolate phosphate trianion intermediate. We report that there is a large ~6 pK unit increase in the basicity of the carboxylate side chain of Glu-167 upon binding of the inhibitor phosphoglycolate trianion (I(3-)), an analog of the enediolate phosphate intermediate, from pKEH ≈ 4 for the protonated free enzyme EH to pK(EHI) ≈ 10 for the protonated enzyme-inhibitor complex EH•I(3-). We propose that there is a similar increase in the basicity of this side chain when the physiological substrates are deprotonated by TbbTIM to form an enediolate phosphate trianion intermediate and that it makes an important contribution to the enzymatic rate acceleration. The affinity of wildtype TbbTIM for I(3-) increases 20,000-fold upon decreasing the pH from 9.3 to 4.9, because TbbTIM exists mainly in the basic form E over this pH range, while the inhibitor binds specifically to the rare protonated enzyme EH. This reflects the large increase in the basicity of the carboxylate side chain of Glu-167 upon binding of I(3-) to EH to give EH•I(3-). The I172A mutation at TbbTIM results in an ~100-fold decrease in the affinity of TbbTIM for I(3-) at pH < 6 and an ~2 pK unit decrease in the basicity of the carboxylate side chain of Glu-167 at the EH•I(3-) complex, to pK(EHI) = 7.7. Therefore, the hydrophobic side chain of Ile-172 plays a critical role in effecting the large increase in the basicity of the catalytic base upon the binding of substrate and/or inhibitors.

Dynamics for the reactions of ion pair intermediates of solvolysis

Publisher Summary This chapter discusses the dynamics for the reactions of ion pair intermediates of solvolysis. It focuses on determining partition rate constant ratios for a variety of reactions of ion pairs, and of absolute rate constants from these ratios. This has been accomplished by use of one of the rate constants from this product ratio as a “clock” for the second reaction. The addition of water to a free carbocation intermediate of solvolysis has been distinguished from addition to an ion-pair intermediate by an examination of common ion inhibition of solvolysis. The observation of “hidden” reactions during solvolysis, through the use of chiral or isotopically labeled substrates has created considerable excitement in communities interested in the mechanisms of non-enzymatic and enzyme catalyzed reactions. The racemization of chiral substrate or the exchange of bridging and nonbridging oxygen during solvolysis may occur through an ion-pair reaction intermediate that is sufficiently long-lived to undergo reorganization in a solvent cage, or it may proceed by an effectively “concerted” mechanism over an energy maximum that closely resembles the carbocation–anion pair intermediate. Another historically important reaction is the reorganization of “chiral” ion pair intermediates of solvolysis of a chiral substrate that leads to racemization of substrate during solvolysis.

Mechanism for Activation of Triosephosphate Isomerase by Phosphite Dianion: The Role of a Hydrophobic Clamp

The role of the hydrophobic side chains of Ile-172 and Leu-232 in catalysis of the reversible isomerization of R-glyceraldehyde 3-phosphate (GAP) to dihydroxyacetone phosphate (DHAP) by triosephosphate isomerase (TIM) from Trypanosoma brucei brucei (Tbb) has been investigated. The I172A and L232A mutations result in 100- and 6-fold decreases in k(cat)/K(m) for the isomerization reaction, respectively. The effect of the mutations on the product distributions for the catalyzed reactions of GAP and of [1-(13)C]-glycolaldehyde ([1-(13)C]-GA) in D(2)O is reported. The 40% yield of DHAP from wild-type Tbb TIM-catalyzed isomerization of GAP with intramolecular transfer of hydrogen is found to decrease to 13% and to 4%, respectively, for the reactions catalyzed by the I172A and L232A mutants. Likewise, the 13% yield of [2-(13)C]-GA from isomerization of [1-(13)C]-GA in D(2)O is found to decrease to 2% and to 1%, respectively, for the reactions catalyzed by the I172A and L232A mutants. The decrease in the yield of the product of intramolecular transfer of hydrogen is consistent with a repositioning of groups at the active site that favors transfer of the substrate-derived hydrogen to the protein or the oxygen anion of the bound intermediate. The I172A and L232A mutations result in (a) a >10-fold decrease (I172A) and a 17-fold increase (L232A) in the second-order rate constant for the TIM-catalyzed reaction of [1-(13)C]-GA in D(2)O, (b) a 170-fold decrease (I172A) and 25-fold increase (L232A) in the third-order rate constant for phosphite dianion (HPO(3)(2-)) activation of the TIM-catalyzed reaction of GA in D(2)O, and (c) a 1.5-fold decrease (I172A) and a larger 16-fold decrease (L232A) in K(d) for activation of TIM by HPO(3)(2-) in D(2)O. The effects of the I172A mutation on the kinetic parameters for the wild-type TIM-catalyzed reactions of the whole substrate and substrate pieces are consistent with a decrease in the basicity of the carboxylate side chain of Glu-167 for the mutant enzyme. The data provide striking evidence that the L232A mutation leads to a ca. 1.7 kcal/mol stabilization of a catalytically active loop-closed form of TIM (E(C)) relative to an inactive open form (E(O)).

Intrinsic barriers to the formation and reaction of carbocations

The Marcus equation relates the activation barriers (AG%) for organic reactions to the overall thermodynamic driving force (AGO) and the intrinsic barrier A, which is the value of AGS when AGO = 0. Most treatments of substituent effects on the rate and equilibrium constants for the reactions of structurally homologous organic substrates make the assumption that the intrinsic barrier A remains constant with changes in AGO. However, there is good evidence for the nucleophilic addition of solvent to carbocations XArCH(R)+ that changes in the aromatic ring substituent X and the benzyl substituent R result in large changes in the intrinsic barrier. Evidence is presented that: (1) There is no significant work required for formation of the reactive complex for proton transfer from carboxylic acids to a homologous series of a-methoxystyrenes XArC(OMe)=CH2. (2) There is a difference in the curvature of the energy surfaces for the reactant and product states that results in their intersection at a transition state in which proton transfer has proceeded ca. two-thirds of the way from RC02H to XArC(OMe)=CH2.

Structural Mutations That Probe the Interactions between the Catalytic and Dianion Activation Sites of Triosephosphate Isomerase

Triosephosphate isomerase (TIM) catalyzes the isomerization of dihydroxyacetone phosphate to form d-glyceraldehyde 3-phosphate. The effects of two structural mutations in TIM on the kinetic parameters for catalysis of the reaction of the truncated substrate glycolaldehyde (GA) and the activation of this reaction by phosphite dianion are reported. The P168A mutation results in similar 50- and 80-fold decreases in (kcat/Km)E and (kcat/Km)E·HPi, respectively, for deprotonation of GA catalyzed by free TIM and by the TIM·HPO(3)(2-) complex. The mutation has little effect on the observed and intrinsic phosphite dianion binding energy or the magnitude of phosphite dianion activation of TIM for catalysis of deprotonation of GA. A loop 7 replacement mutant (L7RM) of TIM from chicken muscle was prepared by substitution of the archaeal sequence 208-TGAG with 208-YGGS. L7RM exhibits a 25-fold decrease in (kcat/Km)E and a larger 170-fold decrease in (kcat/Km)E·HPi for reactions of GA. The mutation has little effect on the observed and intrinsic phosphodianion binding energy and only a modest effect on phosphite dianion activation of TIM. The observation that both the P168A and loop 7 replacement mutations affect mainly the kinetic parameters for TIM-catalyzed deprotonation but result in much smaller changes in the parameters for enzyme activation by phosphite dianion provides support for the conclusion that catalysis of proton transfer and dianion activation of TIM take place at separate, weakly interacting, sites in the protein catalyst.