Evolution in glycolysis

mutagenesis to replace all the wild-type tryptophan residues of the protein, usually by tyrosine. (ii) Measurement of the properties of the tryptophan-less mutant to establish these have not been changed by mutation; (iii) Insertion of a single tryptophan residue a t the site where motion is to be measured. (iv) Characterization of that motion from the changed single-tryptophan fluorescence intensity or anisotropy induced when motion is triggered by substrate-, effectoror inhibitor-binding or by parameters such as temperature, pressure, solvent. viscosity, etc. (v) Repetition of steps ( i i i ) and (iv) with tryptophan probes a t all sites within ii sub-structure until a map of the whole domain motion is completed. A similar approach using chemical modification of a uniquely reactive single inserted amino acid (e.g. cysteine) is also possible. Successively replacing the wild-type tryptophan residues (80. 150. 203) of Bucillus slrurothermophilus LDH by tyrosine did not change substrate or coenzyme-binding, regulation by fructose-l,6-bisphosphate, k,;,, or thermal stability (Waldmnn P I ul.. 1987). The gene lacking tryptophan codons was then used (Clarke i’t ul.. 1986) to generate single tryptophan mutants a t 106 (Gly + Trp), 216 (Lys + Trp), 237 (Tyr + Trp) and 248 (Tyr + Trp). These site-directed mutants had unchanged enzyme properties ~ with the exception of 2 16. Removal of a surface ion pair (Lys-216 to Glu-224) reduces the thermal stability from 90 to 70°C and is consistent with this surface loop (210224) being a hot spot from which thermal folding and unfolding nucleates. Tryphophan-248 is designed to probe the formation of the Q-axis intersubunit contact, Trp-203 to probe for the P-axis intersubunit contact (Clarke C I d., 1 9 8 5 ~ ) and Trp-237 as a probe on the ‘body‘ of the protein for coenzyme loopclosure (Figs. I and 2 ~ ) . The method has been initially tested by mapping the movement of the coenzyme loop (residues 98-1 10) of LDH. The movement by 1.3 nm (defined by the apoand ternary crystal structures) of this polypeptide loop to close over the surface of coenzyme bound to the active ste of LDH is triggered by the entry of pyruvate into the enzymecoenzyme active site. The movement serves two roles: first, i t solvates the bound-nicotinamide coenzyme ring with nonpolar amino acids and is the basis of the oil-water--histidine mechanism of this enzyme (Parker & Holbrook. 1977); more recently Clarke cJt ul. (1986) used site-directed mutagenesis to reveal that loop movement swings Arg-109 into the active centre to polarize the pyruvate carbonyl and to reduce the energy of the transition state. Rates of defined movements have previously been probed: “C-n.m.r. a t Cys-165 revealed a rate equal to the reverse V,,, (200s I at room temperature; Waldman et ul., 1986) while the spectrum of nitrotyrosine237 (Parker et ul., 1982, Clarke et ul., 1985h) revealed a faster rate (3000 s I ) at room temperature. We report the use of one cycle of the new method to probe loop-movement (at position 106) t o produce a n initial threepoint low-resolution map of the domain movement. Fig. 2u shows a super-imposition of the extreme (apoand ternary) crystal structures (from M. G. Rossmann’s laboratory via the Brookhaven Protein Data Bank files) into which are built the gene-derived sequence of the bacterial enzyme (J. J. Holbrook, unpublished work). As expected, internalizing T r p a t 106 gives 15% increased fluorescence intensity owing to reduced solvent collision quenching. The firstorder rate of T r p movement is the same as the V,,, both a t ~ 16°C (Fig. 2h; 2.5 s I ) and a t 25°C (250s I ) . The initial three-point map of domain movement is shown in Fig. 21, and reveals a t least two components to the motion. More mutant probes will give greater kinetic and structural resolution. The decoupling of these two motions provides an explanation for the stabilization a t ~ 16°C in 30% DMSO of half the enzyme in a superactive form (with k,,, 20 times that of the steady state; Clarke rt ul., 19856) in which Arg-109 has already swung into the active centre. The rate of oxidation of N A D H is then only limited by the fast movement sensed by Tyr-237 after pyruvate has collided with the enzyme (Fig. 2 d ) . Under normal turnover conditions recognition of pyruvate by this enzyme involves a t least three steps: bimolecular diffusion into the active site; protein rearrangement around Tyr-237 a t 3000 s I and coenzyme loop closure at 200 s I .