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The Role of Ligand-Driven Conformational Changes in Enzyme Catalysis: Modeling the Reactivity of the Catalytic Cage of Triosephosphate Isomerase Yashraj S. Kulkarni, Qinghua Liao, Fabian Byléhn, Tina L. Amyes, John P. Richard, and Shina C. L. Kamerlin J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b00251 • Publication Date (Web): 08 Mar 2018 Downloaded from http://pubs.acs.org on March 8, 2018
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Journal of the American Chemical Society
The Role of Ligand-Driven Conformational Changes in Enzyme Catalysis: Modeling the Reactivity of the Catalytic Cage of Triosephosphate Isomerase Yashraj S. Kulkarni1, Qinghua Liao1, Fabian Byléhn1,2, Tina L. Amyes3, John P. Richard3,* and Shina C. L. Kamerlin1,* 1
Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, BMC Box 596, S-751 24 Uppsala, Sweden. 2 Department of Chemical Engineering, University College London, Torrington Place, London WC1E 7JE, United Kingdom. 3 Department of Chemistry, University at Buffalo, SUNY, Buffalo, New York 14260-3000, United States, Supporting Information Placeholder
ABSTRACT: We have previously performed empirical valence bond calculations of the kinetic activation barriers, ∆G‡calc, for the deprotonation of complexes between TIM and the whole substrate glyceraldehyde-3phosphate (GAP, Kulkarni et al. J. Am. Chem. Soc. 2017, 139, 10514-10525). We now extend this work to also study the deprotonation of the substrate pieces glycolaldehyde (GA) and GA•HPi [HPi = phosphite dianion]. Our combined calculations provide activation barriers, ∆G‡calc, for the TIM-catalyzed deprotonation of GAP (12.9±0.8 kcal·mol-1), of the substrate piece GA (15.0±2.4 kcal·mol-1), and of the pieces GA•HPi (15.5±3.5 kcal·mol-1). The effect of bound dianion on ∆G‡calc is small (≤ 2.6 kcal·mol-1), in comparison to the much larger 12 kcal·mol-1 and 5.8 kcal·mol-1 intrinsic phosphodianion and phosphite dianion binding energy utilized to stabilize the transition states for TIMcatalyzed deprotonation of GAP and GA•HPi, respectively. This shows that the dianion binding energy is essentially fully expressed at our protein model for the Michaelis complex, where it is utilized to drive an activating change in enzyme conformation. The results represent an example of the synergistic use of results from experiments and calculations to advance our understanding of enzymatic reaction mechanisms.
Triosephosphate isomerase (TIM), orotidine 5'monophosphate decarboxylase (OMPDC), and glycerol 3-phosphate dehydrogenase (GPDH) utilize protein binding interactions with their substrate dianions to drive conformational changes from floppy, inactive, open enzymes to structured, catalytically active cages for the substrate.1 This dianion activation provides the high specificity in transition state binding that is one hallmark of efficient enzymatic catalysis.1c The devel-
opment of an understanding of the mechanism for dianion activation is tightly linked to the broader issue of allosteric activation of enzyme catalysis,2 and the mechanism by which enzymes achieve their high specificity in transition state binding. The latter has been a goal of mechanistic enzymologists since Jencks first described the complexity of this problem in a classic 1975 review.3 Scheme 1. (A) The Mechanism for the TIMCatalyzed Reaction. (B) TIM-Catalyzed Reactions of GAP and the Substrate Pieces GA•HPi. GAP Glu167 E-CO2
+
H
His95 N
O
HN OH
H
H
E
OPO32-
DHAP H E-CO2H
OH
N H
HN
E
O
E-CO2
OPO32-
P
(
E
H OH
+
N HN
O
E
OPO32-
) GA /K m
k cat
B
N HN
OH
OPO32-
A
O
E-CO2H E
E • GAP
+ GA + HPi [ΔG‡GAP]
± GAP ± GA + HPi [ΔG‡
kc
at /K HP
iK GA
ΔΔG‡ Products
GA+HPi]
+ GAP
GA E • HPi
TIM catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to (R)-glyceraldehyde 3-phosphate (GAP), through enzyme-bound cis-enediolate reaction intermediates (Scheme 1A),4 and phosphite dianionactivated deprotonation of glycolaldehyde (GA).5 The kinetic parameters, kcat/Km (M-1 s-1), for the TIMcatalyzed isomerization of the whole substrate GAP to form DHAP, and kcat/KGAKHPi (M-2 s-1) for phosphite dianion activated reactions of the substrate piece GA 1
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(Scheme 1B), determined for fourteen different wildtype and site-directed mutant forms of TIM, define an extraordinary linear free energy relationship (LFER), with slope of 1.0, between the activation barriers DG‡ for the wildtype and mutant enzyme-catalyzed reactions of the whole substrate and substrate pieces.6 The LFER is consistent with Scheme 2,6a which shows dianion activators as functioning exclusively to stabilize the active closed form of TIM (EC).1c, 5a This closed form exists at low concentrations in the absence of the activating dianion (KC
