Figure 1. Proposed enzymatic mechanism of the α-reaction of TRPS. B2 likely is αAsp60, while αGlu49 is believed to have a dual role as proton acceptor (B1H) and donor (B3).

Figure 2. (a) 2-Aminophenol functions as an analogue of indole and reacts covalently with G3P at the α-site of TPRS. There are two possible adducts, either (b) 1-[2-hydroxyphenylamino]3-glycerolphosphate or (c) 1-2-aminophenoxy3-glycerolphosphate.

Figure 3. The crystal structure of TRPS complexed with the transition state analogue reveals interactions between the substrate and catalytically important residues. The σ_A-weighted 2F_obs−F_calc electron density is shown at a contour level of 1.0σ for the transition state analogue, 2-AP-glycerol phosphate, and for nearby residues in the TRPS-2-amino structure. The side-chain of αGlu49 adopts the active conformation and forms two well-defined hydrogen bonds, fully consistent with the hypothesis that αGlu49 serves both as B1-H and as B3 in the α-reaction mechanism (see Figure 1). The second catalytic residue, αAsp60 (proposed to be B2), interacts with the hydroxyl group of 2-AP-glycerol phosphate (O7) (2.6 Å) and αThr183-OG1 (2.8 Å).

Figure 4. Binding mode of IPG (yellow) and 2-AP-glycerol phosphate (cyan) to the active site of TRPS. There are two conformations of the catalytic αGlu49 in the IGP complex.

Figure 5. (a) Topology of BX1. BX1 has a TIM barrel fold. The N-terminal helix H0 (yellow) is predicted to be a transit peptide; it is not present in cloned protein. The helices H0′, H2′, H8′ (red) are found in BX1 and TRPS-alpha but not in TIM. (b) C^α-atom superposition of the α-subunit of TRPS (yellow) and BX1 (grey). Major structural differences occur at the loop regions. They coincide with the αβ interface region of the α-subunit of TRPS that binds the β-subunit. IPP is shown in stick mode.

Figure 6. Environment of the catalytic glutamate. (a) In TPRS, αGlu49 has two conformations, an extended active one interacting with the 3′-hydroxyl of IGP and an inactive one with the carboxylate hydrogen bonded with αTyr173. (b) In BX1 (yellow), Glu134 is positioned in the active conformation. Due to a number of substitutions, including Phe253↔αTyr173 and Ile207↔αLeu127, the corresponding inactive conformation seen in TPRS is not energetically favorable in the BX1 structure.

Figure 7. The conformation of loop αL6 is determined by the position of an arginine residue. (a) In α-TRPS, the guanidinium group of αArg179 lies in the plane of the loop and forms a number of radial interactions. (b) In BX1 the corresponding guanidinium group, Arg266, is tilted out of the ring plane, resulting in only one interaction with the loop. The two guanidinium groups occupy similar positions in space.

Scheme 1. (a) The free α-subunit is postulated to exist as an ensemble of stable, inactive conformations (α) in equilibrium with the unstable active conformation (α*). (b) Free α-subunit is activated by 30 to 100-fold by binding with the β-subunit to form E(Ain). (c) Reaction of l-Ser with E(Ain) to give E(A-A) stabilizes the α* conformation and gives an additional >30-fold activation. The overall activation of the α-subunit on conversion of the free α-subunit to E(A-A) is at least 1000 to 3000-fold, consequently, the fraction of free α-subunit in the form of α* must be less than 1/3000 to 1/1000. For simplicity, an αβ-dimeric unit of the tetrameric TRPS is shown in (b) and (c).

Crystal parameters, data collection, and refinement statistics

R_sym=∑|I−I|/∑I

Apparent dissociation constants, K_Dapp, from the fluorescence titration of wild-type enzyme with 2AP and G3P

Reaction conditions: [enzyme sites], 10 μM; [ANS], 13 μM and [NaCl], 100 mM. Reactions were measured at 25 °C in 50 mM TEA buffer (pH 7.8).