Enabling Role of Ligand-Driven Conformational Changes in Enzyme Evolution

Abstract

Many enzymes that show a large specificity in binding the enzymatic transition state with a higher affinity than the substrate utilize substrate binding energy to drive protein conformational changes to form caged substrate complexes. These protein cages provide strong stabilization of enzymatic transition states. Using part of the substrate binding energy to drive the protein conformational change avoids a similar strong stabilization of the Michaelis complex and irreversible ligand binding. A seminal step in the development of modern enzyme catalysts was the evolution of enzymes that couple substrate binding to a conformational change. These include enzymes that function in glycolysis (triosephosphate isomerase), the biosynthesis of lipids (glycerol phosphate dehydrogenase), the hexose monophosphate shunt (6-phosphogluconate dehydrogenase), and the mevalonate pathway (isopentenyl diphosphate isomerase), catalyze the final step in the biosynthesis of pyrimidine nucleotides (orotidine monophosphate decarboxylase), and regulate the cellular levels of adenine nucleotides (adenylate kinase). The evolution of enzymes that undergo ligand-driven conformational changes to form active protein−substrate cages is proposed to proceed by selection of variants, in which the selected side chain substitutions destabilize a second protein conformer that shows compensating enhanced binding interactions with the substrate. The advantages inherent to enzymes that incorporate a conformational change into the catalytic cycle provide a strong driving force for the evolution of flexible protein folds such as the TIM barrel. The appearance of these folds represented a watershed event in enzyme evolution that enabled the rapid propagation of enzyme activities within enzyme superfamilies.

Adenylate Kinase-Catalyzed Reactions of AMP in Pieces: Specificity for Catalysis at the Nucleoside Activator and Dianion Catalytic Sites

“…With this in mind, we targeted adenylate kinase for study, to test our hypothesis that the pressure to optimize the enzymatic rate acceleration led to the utilization of protein−adenosyl group binding interactions to drive a protein conformational change, which activates the enzyme for catalysis of phosphoryl transfer. 10 This hypothesis was supported by our recent report that: (1) the binding interactions between human adenylate kinase isozyme 1 (HAdK1) and the adenosyl group of substrate AMP provide a ≥14.7 kcal mol −1 stabilization of the transition state for phosphoryl transfer from ATP to AMP (Scheme 1). 23 (2) Greater than 8.7 kcal mol −1 of this transition state stabilization is recovered, after severing the covalent connection between the adenosyl and phosphate substrate fragments of AMP, as activation of HAdK1-catalyzed phosphoryl transfer from ATP to the substrate piece phosphite dianion by the second nonreacting piece 1-(β-D-erythrofuranosyl)adenine (EA) (Scheme 3).…”

“…39 The difference between the small observed binding energy for the substrates OMP and AMP, respectively, of reactions catalyzed by OMPDC and adenylate kinase; and, their much larger intrinsic transition state binding energies represents, in part, the substrate binding energy utilized to drive unfavorable enzyme conformational changes (ΔG C , Scheme 2). 10 It is important to emphasize that this binding energy may also be used to induce destabilizing electrostatic stress between protein side chains and OMP bound to OMPDC 12,18,40 or between the terminal phosphate dianions of ATP and AMP bound to adenylate kinase that is relieved at the enzymatic transition states. 18 There is evidence that the former destabilizing interactions are small for OMPDC-catalyzed decarboxylation, 41,42 but the binding energy utilized to introduce electrostatic stress at the ternary Michaelis complex for adenylate kinase has not been evaluated.…”

“…52,53 We have concluded that the phosphodianion-driven loop closure at OMPDC has the effect of placing the protein side chains at the orotate ring at positions that provide optimal stabilization of the decarboxylation transition state. 6,7,10,11 The 11.8 kcal mol −1 adenine ring binding energy (Figure 6) is utilized to drive an enzyme conformational change, 3,4 which places the protein side chains at positions around the bound ATP and AMP substrates that provide for optimal stabilization of the transition state for enzyme-catalyzed phosphoryl transfer. The side chains and other structural elements that function to drive this conformational change have not yet been identified.…”

“…Enzymes that catalyze a diverse set of proton transfer, 13,14 hydride transfer, 14,15 and decarboxylation 14,16 reactions of phosphorylated substrates use the intrinsic phosphodianion binding energy to drive thermodynamically unfavorable protein conformational changes. 10,11,17 The dianion binding energy so utilized is not expressed at the Michaelis complex, but is only expressed at the transition state and provides for specificity in binding of enzymatic transition states with a higher affinity than substrate. 10−12 This is often required to avoid tight and effectively irreversible substrate binding.…”

“…The induced-fit mechanism was proposed more than 60 years ago to rationalize enzyme specificity in the catalysis of reactions of substrates that provide binding energy to drive a protein conformational change (Scheme ). , We have shown that such substrate-driven conformational changes are the defining property of many enzymes that follow this mechanism, − where the use of substrate binding energy in the formation of the Michaelis complex ensures that the enzymatic transition state binds with a higher affinity than substrate.…”