Enzyme Architecture: Breaking Down the Catalytic Cage that Activates Orotidine 5′-Monophosphate Decarboxylase for Catalysis

The enormous rate accelerations observed for many enzyme catalysts are due to strong stabilizing interactions between the protein and reaction transition state. The defining property of these catalysts is their specificity for binding the transition state with a much higher affinity than substrate. Experimental results are presented which show that the phosphodianion-binding energy of phosphate monoester substrates is used to drive conversion of their protein catalysts from flexible and entropically rich ground states to stiff and catalytically active Michaelis complexes. These results are generalized to other enzyme-catalyzed reactions. The existence of many enzymes in flexible, entropically rich, and inactive ground states provides a mechanism for utilization of ligand-binding energy to mold these catalysts into stiff and active forms. This reduces the substrate-binding energy expressed at the Michaelis complex, while enabling the full and specific expression of large transition-state binding energies. Evidence is presented that the complexity of enzyme conformational changes increases with increases in the enzymatic rate acceleration. The requirement that a large fraction of the total substrate-binding energy be utilized to drive conformational changes of floppy enzymes is proposed to favor the selection and evolution of protein folds with multiple flexible unstructured loops, such as the TIM-barrel fold. The effect of protein motions on the kinetic parameters for enzymes that undergo ligand-driven conformational changes is considered. The results of computational studies to model the complex ligand-driven conformational change in catalysis by triosephosphate isomerase are presented.

show abstract

“…The blue loops interact at the closed form of OMPDC through a hydrogen bond between the side chains of S154 and Q215. 76 , 77 …”

Section: A Role For Protein Conformational Changes In Enzyme Catalysimentioning

confidence: 99%

Protein Flexibility and Stiffness Enable Efficient Enzymatic Catalysis

2019

Self Cite

View full text Add to dashboard Cite

show abstract

“… 119,120 This is the case of triosephosphate isomerase, 121 glycerol phosphate dehydrogenase 122 and orotidine 5'-monophosphate decarboxylase. 123 However, the kinetic mechanism of catalysis also requires an inactive open form of the enzyme to coexist in equilibrium with the active closed form prior to ligand binding, as envisioned by CS. 119 Selective binding of the ligand to the active form is then the trigger for expression of the full transition-state binding energy.…”

Section: Distinguishing Between If and Csmentioning

confidence: 99%

Mechanisms of ligand binding

2020

Biophysics Reviews

View full text Add to dashboard Cite

Many processes in chemistry and biology involve interactions of a ligand with its molecular target. Interest in the mechanism governing such interactions has dominated theoretical and experimental analysis for over a century. The interpretation of molecular recognition has evolved from a simple rigid body association of the ligand with its target to appreciation of the key role played by conformational transitions. Two conceptually distinct descriptions have had a profound impact on our understanding of mechanisms of ligand binding. The first description, referred to as induced fit, assumes that conformational changes follow the initial binding step to optimize the complex between the ligand and its target. The second description, referred to as conformational selection, assumes that the free target exists in multiple conformations in equilibrium and that the ligand selects the optimal one for binding. Both descriptions can be merged into more complex reaction schemes that better describe the functional repertoire of macromolecular systems. This review deals with basic mechanisms of ligand binding, with special emphasis on induced fit, conformational selection, and their mathematical foundations to provide rigorous context for the analysis and interpretation of experimental data. We show that conformational selection is a surprisingly versatile mechanism that includes induced fit as a mathematical special case and even captures kinetic properties of more complex reaction schemes. These features make conformational selection a dominant mechanism of molecular recognition in biology, consistent with the rich conformational landscape accessible to biological macromolecules being unraveled by structural biology.

show abstract

“…Apparent first-order rate constants k obs (s –1 ) for the T100′A ([E] = 130 nM, monitored at 282 nm) and T100′G ([E] = 35 μM, monitored at 292 nm) variants of the OMPDC-catalyzed decarboxylations of FOMP at 25 °C, pH 7.1 (30 mM MOPS), and I = 0.105 (NaCl) were determined from the fit to an exponential decay over at least 5 reaction half-life times at [FOMP] ≤ 0.1 K m . 18 The second-order rate constant, k cat / K m (M –1 s –1 ), for the variant OMPDC-catalyzed decarboxylation of FOMP was calculated from the relationship k cat / K m = k obs /[E].…”

Section: Methodsmentioning

confidence: 99%

“… 16 − 20 These side chain interactions provide a significant fraction of the driving force that activates the ligand-driven change in the enzyme conformation ( Scheme 3 ), which results in the immobilization of two flexible protein loops by interactions with the substrate dianion and the pyrimidine ring. 18 , 21 …”

mentioning

confidence: 99%

Orotidine 5′-Monophosphate Decarboxylase: The Operation of Active Site Chains Within and Across Protein Subunits

Brandão

Richard

2020

Biochemistry

Self Cite

View full text Add to dashboard Cite

The D37 and T100′ side chains of orotidine 5′-monophosphate decarboxylase (OMPDC) interact with the C-3′ and C-2′ ribosyl hydroxyl groups, respectively, of the bound substrate. We compare the intra-subunit interactions of D37 with the inter-subunit interactions of T100′ by determining the effects of the D37G, D37A, T100′G, and T100′A substitutions on the following: (a) k cat and k cat / K m values for the OMPDC-catalyzed decarboxylations of OMP and 5-fluoroorotidine 5′-monophosphate (FOMP) and (b) the stability of dimeric OMPDC relative to the monomer. The D37G and T100′A substitutions resulted in 2 kcal mol –1 increases in Δ G † for k cat / K m for the decarboxylation of OMP, while the D37A and T100′G substitutions resulted in larger 4 and 5 kcal mol –1 increases, respectively, in Δ G † . The D37G and T100′A substitutions both resulted in smaller 2 kcal mol –1 decreases in Δ G † for the decarboxylation of FOMP compared to that of OMP. These results show that the D37G and T100′A substitutions affect the barrier to the chemical decarboxylation step while the D37A and T100′G substitutions also affect the barrier to a slow, ligand-driven enzyme conformational change. Substrate binding induces the movement of an α-helix (G′98–S′106) toward the substrate C-2′ ribosyl hydroxy bound at the main subunit. The T100′G substitution destabilizes the enzyme dimer by 3.5 kcal mol –1 compared to the monomer, which is consistent with the known destabilization of α-helices by the internal Gly side chains [Serrano, L., et al. (1992) Nature , 356 , 453–455]. We propose that the T100′G substitution weakens the α-helical contacts at the dimer interface, which results in a decrease in the dimer stability and an increase in the barrier to the ligand-driven conformational change.

show abstract

Enzyme Architecture: Breaking Down the Catalytic Cage that Activates Orotidine 5′-Monophosphate Decarboxylase for Catalysis

Cited by 12 publications

References 47 publications

Protein Flexibility and Stiffness Enable Efficient Enzymatic Catalysis

Protein Flexibility and Stiffness Enable Efficient Enzymatic Catalysis

Mechanisms of ligand binding

Orotidine 5′-Monophosphate Decarboxylase: The Operation of Active Site Chains Within and Across Protein Subunits

Contact Info

Product

Resources

About