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Enzyme activation through the utilization of intrinsic dianion binding energy.

ABSTRACT: 43:We consider 'the proposition that the intrinsic binding energy that results from the noncovalent interaction of a specific substrate with the active site of the enzyme is considerably larger than is generally believed. An important part of this binding energy may be utilized to provide the driving force for catalysis, so that the observed binding energy represents only what is left over after this utilization' [Jencks,W.P. (1975) Adv. Enzymol. Relat. Areas. Mol. Biol. , , 219-410]. The large ~12 kcal/mol intrinsic substrate phosphodianion binding energy for reactions catalyzed by triosephosphate isomerase (TIM), orotidine 5'-monophosphate decarboxylase and glycerol-3-phosphate dehydrogenase is divided into 4-6 kcal/mol binding energy that is expressed on the formation of the Michaelis complex in anchoring substrates to the respective enzyme, and 6-8 kcal/mol binding energy that is specifically expressed at the transition state in activating the respective enzymes for catalysis. A structure-based mechanism is described where the dianion binding energy drives a conformational change that activates these enzymes for catalysis. Phosphite dianion plays the active role of holding TIM in a high-energy closed active form, but acts as passive spectator in showing no effect on transition-state structure. The result of studies on mutant enzymes is presented, which support the proposal that the dianion-driven enzyme conformational change plays a role in enhancing the basicity of side chain of E167, the catalytic base, by clamping the base between a pair of hydrophobic side chains. The insight these results provide into the architecture of enzyme active sites and the development of strategies for the de novo design of protein catalysts is discussed.

SUBMITTER: Amyes TL

PROVIDER: S-EPMC6388876 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Enzyme activation through the utilization of intrinsic dianion binding energy.

Amyes T L TL Malabanan M M MM Zhai X X Reyes A C AC Richard J P JP

Protein engineering, design & selection : PEDS 20170301 3

<h4>43</h4>We consider 'the proposition that the intrinsic binding energy that results from the noncovalent interaction of a specific substrate with the active site of the enzyme is considerably larger than is generally believed. An important part of this binding energy may be utilized to provide the driving force for catalysis, so that the observed binding energy represents only what is left over after this utilization' [Jencks,W.P. (1975) Adv. Enzymol. Relat. Areas. Mol. Biol. , , 219-410]. Th ...[more]

PMID: 27903763

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Project description:BACKGROUND: Models that are capable of reliably predicting binding affinities for protein-ligand complexes play an important role the field of structure-guided drug design. METHODS: Here, we begin by applying the computational geometry technique of Delaunay tessellation to each set of atomic coordinates for over 1400 diverse macromolecular structures, for the purpose of deriving a four-body statistical potential that serves as a topological scoring function. Next, we identify a second, independent set of three hundred protein-ligand complexes, having both high-resolution structures and known dissociation constants. Two-thirds of these complexes are randomly selected to train a predictive model of binding affinity as follows: two tessellations are generated in each case, one for the entire complex and another strictly for the isolated protein without its bound ligand, and a topological score is computed for each tessellation with the four-body potential. Predicted protein-ligand binding affinity is then based on an empirically derived linear function of the difference between both topological scores, one that appropriately scales the value of this difference. RESULTS: A comparison between experimental and calculated binding affinity values over the two hundred complexes reveals a Pearson's correlation coefficient of r = 0.79 with a standard error of SE = 1.98 kcal/mol. To validate the method, we similarly generated two tessellations for each of the remaining protein-ligand complexes, computed their topological scores and the difference between the two scores for each complex, and applied the previously derived linear transformation of this topological score difference to predict binding affinities. For these one hundred complexes, we again observe a correlation of r = 0.79 (SE = 1.93 kcal/mol) between known and calculated binding affinities. Applying our model to an independent test set of high-resolution structures for three hundred diverse enzyme-inhibitor complexes, each with an experimentally known inhibition constant, also yields a correlation of r = 0.79 (SE = 2.39 kcal/mol) between experimental and calculated binding energies. CONCLUSIONS: Lastly, we generate predictions with our model on a diverse test set of one hundred protein-ligand complexes previously used to benchmark 15 related methods, and our correlation of r = 0.66 between the calculated and experimental binding energies for this dataset exceeds those of the other approaches. Compared with these related prediction methods, our approach stands out based on salient features that include the reliability of our model, combined with the rapidity of the generated predictions, which are less than one second for an average sized complex.

Project description: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)).

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Enzyme activation through the utilization of intrinsic dianion binding energy.

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Enzyme activation through the utilization of intrinsic dianion binding energy.

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