A role for flexible loops in enzyme catalysis

Abstract

Triosephosphate isomerase (TIM), glycerol 3-phosphate dehydrogenase, and orotidine 5'-monophosphate decarboxylase each use the binding energy from the interaction of phosphite dianion with a flexible phosphate gripper loop to activate a second, phosphodianion-truncated, substrate towards enzyme-catalyzed proton transfer, hydride transfer, and decarboxylation, respectively. Studies on TIM suggest that the most important general effect of loop closure over the substrate phosphodianion, and the associated conformational changes, is to extrude water from the enzyme active site. This should cause a decrease in the effective active-site dielectric constant, and an increase in transition state stabilization from enhanced electrostatic interactions with polar amino acid side chains. The most important specific effect of these conformational changes is to increase the basicity of the carboxylate side chain of the active site glutamate base by its placement in a 'hydrophobic cage'.

Figure 1

(A) The isomerization of GAP to DHAP catalyzed by TIM, which proceeds by a proton transfer mechanism through an enediolate intermediate [4,8]. (B) The crystal structures of the loop-open and loop-closed forms of TIM from chicken muscle. The aqua ribbons are for the open unliganded enzyme [PDB entry 1TIM] [17] and the green ribbons show the closed enzyme liganded with 2-phosphoglycolohydroxamate (PGH) [PDB entry 1TPH] [13].

Figure 1

(A) The isomerization of GAP to DHAP catalyzed by TIM, which proceeds by a proton transfer mechanism through an enediolate intermediate [4,8]. (B) The crystal structures of the loop-open and loop-closed forms of TIM from chicken muscle. The aqua ribbons are for the open unliganded enzyme [PDB entry 1TIM] [17] and the green ribbons show the closed enzyme liganded with 2-phosphoglycolohydroxamate (PGH) [PDB entry 1TPH] [13].

Figure 2

(A) Decarboxylation reaction catalyzed by orotidine 5'-monophosphate decarboxylase. (B) Hydride transfer reaction catalyzed by glycerol 3-phosphate dehydrogenase.

Chart 1

A comparison of the intrinsic binding energies (IBEs) of the substrate phosphodianion group and the phosphite dianion piece for the reactions catalyzed by OMPDC, TIM, and GPDH. The IBE for the substrate phosphodianion is calculated from the ratio of the second-order rate constants for turnover of the whole [(k_cat/K_m)_SPi] and the truncated [(k_cat/K_m)_S] substrates. The IBE for phosphite dianion is calculated from the ratio of the third-order rate constant for the phosphite-activated turnover of the truncated substrate [(k_cat/K_m)_S/K_d] and the second-order rate constant for turnover of the truncated substrate [(k_cat/K_m)_S].

Figure 3

Space-filling model of the surface of yeast TIM, with DHAP bound, in the region of the active site [PDB entry 1NEY] [9]. The flexible loop 6 is colored cyan, the alkylammonium side chain of Lys-12 is colored black (C), white (H) and blue (N). The visible part of the bound substrate is colored red (O) and orange (P).

Figure 4

The highly conserved [44,45] sequence of loop 6 of TIM from chicken muscle.

Figure 5

A comparison of the relaxed open form and the tightened closed form of TIM from chicken muscle showing the formation of important inter-loop interactions upon ligand binding. (A) The active site of the unliganded open conformation [PDB entry 1TIM] [17]. (B) The active site of TIM liganded with PGH [PDB entry 1TPH] [13], an analog of the enediolate intermediate [12].

Figure 5

A comparison of the relaxed open form and the tightened closed form of TIM from chicken muscle showing the formation of important inter-loop interactions upon ligand binding. (A) The active site of the unliganded open conformation [PDB entry 1TIM] [17]. (B) The active site of TIM liganded with PGH [PDB entry 1TPH] [13], an analog of the enediolate intermediate [12].

Figure 6

The 0.82 Å resolution structure of the complex between TIM (L. mexicana) and the enediolate intermediate analog PGH [PDB entry 2VXN] [12]. PGH is bound in the planar amidate form [12,54] and it was suggested that the N-H of PGH lies closest to the carboxylate oxygen [12]. The distances from N-2 of His-95 to O-1 (2.8 Å) and O-2 (2.8 Å) of PGH are as expected for normal hydrogen bonds [12]. The inset compares the structures of the amidate form of PGH and the enediolate intermediate of the TIM-catalyzed isomerization reaction.

Figure 6

The 0.82 Å resolution structure of the complex between TIM (L. mexicana) and the enediolate intermediate analog PGH [PDB entry 2VXN] [12]. PGH is bound in the planar amidate form [12,54] and it was suggested that the N-H of PGH lies closest to the carboxylate oxygen [12]. The distances from N-2 of His-95 to O-1 (2.8 Å) and O-2 (2.8 Å) of PGH are as expected for normal hydrogen bonds [12]. The inset compares the structures of the amidate form of PGH and the enediolate intermediate of the TIM-catalyzed isomerization reaction.

Figure 7

(A) The structure of the complex between TIM (T. brucei) and glycerol 3-phosphate (G3P) [PDB entry 6TIM] [58]. The side chain of Glu-167 sits in a “hydrophobic cage”. (B) The structure of I172A mutant TIM (T. brucei) complexed with G3P, prepared starting with the coordinates for the wildtype enzyme complexed with G3P [PDB entry 6TIM] [58] and using the molecular graphics software SYBYL version 7.3 (Tripos Inc., St. Louis, MO).

Figure 7

(A) The structure of the complex between TIM (T. brucei) and glycerol 3-phosphate (G3P) [PDB entry 6TIM] [58]. The side chain of Glu-167 sits in a “hydrophobic cage”. (B) The structure of I172A mutant TIM (T. brucei) complexed with G3P, prepared starting with the coordinates for the wildtype enzyme complexed with G3P [PDB entry 6TIM] [58] and using the molecular graphics software SYBYL version 7.3 (Tripos Inc., St. Louis, MO).

Figure 8

A model developed to rationalize the observation that the binding of HPO₃^2- activates enzymes that utilize a flexible phosphate gripper loop for catalysis of the reaction of a truncated substrate S [20]. The enzyme is shown to exist in an inactive loop-open form (E_o) and a rare active loop-closed form (E_c) that is stabilized by the binding of HPO₃^2- [16, 60].