We report that the K12G mutation at triosephosphate isomerase (TIM) from Saccharomyces cerevisiae results in: (1) A ca. 50-fold increase in K m for the substrate glyceraldehyde 3-phosphate (GAP) and a 60-fold increase in K i for competitive inhibition by the intermediate analog 2-phosphoglycolate, resulting from the loss of stabilizing ground state interactions between the alkylammonium side chain of Lys-12 and the ligand phosphodianion group. (2) A 12,000-fold decrease in k cat for isomerization of GAP, suggesting a tightening of interactions between the side chain of Lys-12 and the substrate on proceeding from the Michaelis complex to the transition state. (3) A 6 × 10 5 -fold decrease in k cat /K m , corresponding to a total 7.8 kcal/mol stabilization of the transition state by the cationic side chain of Lys-12. The yields of the four products of the K12G TIM-catalyzed isomerization of GAP in D 2 O were quantified as: dihydroxyacetone phosphate (DHAP), 27%; [1(R)-2 H]-DHAP, 23%; [2(R)-2 H]-GAP, 31%; and 18% methylglyoxal from an enzyme-catalyzed elimination reaction. The K12G mutation has only a small effect on the relative yields of the three products of proton transfer to the TIM-bound enediol(ate) intermediate in D 2 O, but it strongly favors catalysis of the elimination reaction to give methylglyoxal. The K12G mutation also results in a ≥ 14-fold decrease in k cat /K m for isomerization of bound glycolaldehyde (GA), although the dominant observed product of the mutant enzyme-catalyzed reaction of [1-13 C]-GA in D 2 O is [1-13 C, 2,2-di-2 H]-GA from a nonspecific protein-catalyzed reaction. The observation that the K12G mutation results in a large decrease in k cat /K m for the reactions of both GAP and the neutral truncated substrate [1-13 C]-GA provides evidence for a stabilizing interaction between the cationic side chain of Lys-12 and negative charge that develops at the enolate-like oxygen in the transition state for deprotonation of the sugar substrate "piece".Triosephosphate isomerase (TIM) 1 catalyzes the stereospecific, reversible, 1,2-hydrogen shift at dihydroxyacetone phosphate (DHAP) to give (R)-glyceraldehyde 3-phosphate (GAP) by a single-base (Glu-165) proton transfer mechanism through an enzyme-bound cisenediol(ate) intermediate (Scheme 1) (1,2). The enzyme's low molecular weight (dimer, 26 kDa/subunit), high cellular abundance (3), and the centrality of proton transfer at carbon in metabolic processes (4-6) have made TIM a prominent target for studies on the mechanism of enzyme action (1,7-10).

The side chain cation of R269 lies at the surface of l-glycerol 3-phosphate dehydrogenase (GPDH) and forms an ion pair to the phosphodianion of substrate dihydroxyacetone phosphate (DHAP), which is buried at the nonpolar protein interior. The R269A mutation of GPDH results in a 110-fold increase in Km (2.8 kcal/mol effect) and a 41 000-fold decrease in kcat (6.3 kcal/mol effect), which corresponds to a 9.1 kcal/mol destabilization of the transition state for GPDH-catalyzed reduction of DHAP by NADH. There is a 6.7 kcal/mol stabilization of the transition state for the R269A mutant GPDH-catalyzed reaction by 1.0 M guanidinium ion, and the transition state for the reaction of the substrate pieces is stabilized by an additional 2.4 kcal/mol by their covalent attachment at wildtype GPDH. These results provide strong support for the proposal that GPDH invests the 11 kcal/mol intrinsic phosphodianion binding energy of DHAP in trapping the substrate at a nonpolar active site, where strong electrostatic interactions are favored, and obtains a 9 kcal/mol return from stabilizing interactions between the side chain cation and transition state trianion. We propose a wide propagation for the catalytic motif examined in this work, which enables strong transition state stabilization from enzyme–phosphodianion pairs.

The role of the hydrophobic side chains of Ile-172 and Leu-232 in catalysis of the reversible isomerization of R-glyceraldehyde 3-phosphate (GAP) to dihydroxyacetone phosphate (DHAP) by triosephosphate isomerase (TIM) from Trypanosoma brucei brucei (Tbb) has been investigated. The I172A and L232A mutations result in 100- and 6-fold decreases in kcat/Km for the isomerization reaction, respectively. The effect of the mutations on the product distributions for the catalyzed reactions of GAP and of [1-13C]-glycolaldehyde ([1-13C]-GA) in D2O is reported. The 40% yield of DHAP from wildtype TbbTIM-catalyzed isomerization of GAP with intramolecular transfer of hydrogen is found to decrease to 13% and to 4%, respectively, for the reactions catalyzed by the I172A and L232A mutants. Likewise, the 13% yield of [2-13C]-GA from isomerization of [1-13C]-GA in D2O is found to decrease to 2% and to 1%, respectively, for the reactions catalyzed by the I172A and L232A mutants. The decrease in the yield of the product of intramolecular transfer of hydrogen is consistent with a repositioning of groups at the active site that favors transfer of the substrate-derived hydrogen to the protein or the oxygen anion of the bound intermediate. The I172A and L232A mutations result in: (a) A >10-fold decrease (I172A) and a 17-fold increase (L232A) in the second-order rate constant for TIM-catalyzed reaction of [1-13C]-GA in D2O. (b) A 170-fold decrease (I172A) and 25-fold increase (L232A) in the third-order rate constant for phosphite dianion (HPO32-) activation of TIM-catalyzed reaction of GA in D2O. (c) A 1.5-fold decrease (I172A) and a larger 16-fold decrease (L232A) in Kd for activation of TIM by HPO32- in D2O. The effects of the I172A mutation on the kinetic parameters for wildtype TIM-catalyzed reactions of the whole substrate and substrate pieces are consistent with a decrease in the basicity of the carboxylate side chain of Glu-167 for the mutant enzyme. The data provide striking evidence that the L232A mutation leads to a ca. 1.7 kcal/mol stabilization of a catalytically active loop-closed form of TIM (EC) relative to an inactive open form (EO).

The genes encoding Shiga toxin (stx), the major virulence factor of Shiga toxin-encoding Escherichia coli (STEC) strains, are carried on lambdoid prophages resident in all known STEC strains. The stx genes are expressed only during lytic growth of these temperate bacteriophages. We cloned the gene encoding the repressor of the Shiga toxin-encoding bacteriophage 933W and examined the DNA binding and transcriptional regulatory activities of the overexpressed, purified protein. Typical of nearly all lambdoid phage repressors, 933W repressor binds to three sites in 933W right operator (O R ). Also typical, when bound at O R , 933W repressor functions as an activator at the P RM promoter and a repressor at the P R promoter. In contrast to other lambdoid bacteriophages, 933W left operator (O L ) contains only two repressor binding sites, but the O L -bound repressor still efficiently represses P L transcription. Lambdoid prophage induction requires inactivation of the repressor's DNA binding activity. In all phages examined thus far, this inactivation requires a RecA-stimulated repressor autoproteolysis event, with cleavage occurring precisely in an Ala-Gly dipeptide sequence that is found within a "linker " region that joins the two domains of these proteins. However, 933W repressor protein contains neither an Ala-Gly nor an alternative Cys-Gly dipeptide cleavage site anywhere in its linker sequence. We show here that the autocleavage occurs at a Leu-Gly dipeptide. Thus, the specificity of the repressor autocleavage site is more variable than thought previously.Shiga toxins (stx) are the major virulence factors in enterohemorrhagic Escherichia coli infections, causing such diseases as hemorrhagic colitis, infantile diarrhea, and hemolytic uremic syndrome. In virtually all known Shiga toxin-encoding E. coli strains, the genes encoding Shiga toxins are carried on lambdoid prophages (8,19,31,34,35,45) as part of an operon whose activity is ultimately regulated by the bacteriophage repressor (32,33,46,47).Lambdoid phage genomes contain two operator regions, O L and O R , each of which includes promoters whose expression is controlled by the binding of the bacteriophage repressor to multiple binding sites found in each operator region. Efficient functioning of the genetic switch between lysis and lysogeny depends on the ability of the repressor to bind with different affinities to each of the individual sites within O R and O L (40). The repressor directs the establishment and maintenance of the lysogenic state by simultaneously repressing transcription of the genes needed for lytic phage growth and activating transcription of its own gene, the only gene needed for maintenance of the lysogenic state (40).The E. coli O157:H7 strain EDL933 is considered to be the reference strain for disease-causing O157:H7 isolates. Incubating this strain with agents that trigger induction of resident prophages causes this strain to express the disease-causing stx2 gene product and to produce a lambdoid bacteriophage (38). Sequence analysi...

We report the results of a study of the catalytic role of a network of four interacting amino acid side chains at yeast orotidine 5′-monophosphate decarboxylase (ScOMPDC), by the stepwise replacement of all four side chains. The H-bond, which links the −CH2OH side chain of S154 from the pyrimidine umbrella loop of ScOMPDC to the amide side chain of Q215 in the phosphodianion gripper loop, creates a protein cage for the substrate OMP. The role of this interaction in optimizing transition state stabilization from the dianion gripper side chains Q215, Y217, and R235 was probed by determining the kinetic parameter kcat/Km for 16 enzyme variants, which include all combinations of single, double, triple, and quadruple S154A, Q215A, Y217F, and R235A mutations. The effects of consecutive Q215A, Y217F, and R235A mutations on ΔG⧧ for wild-type enzyme-catalyzed decarboxylation sum to 11.6 kcal/mol, but to only 7.6 kcal/mol when starting from S154A mutant. This shows that the S154A mutation results in a (11.6–7.6) = 4.0 kcal/mol decrease in transition state stabilization from interactions with Q215, Y217, and R235. Mutant cycles show that ca. 2 kcal/mol of this 4 kcal/mol effect is from the direct interaction between the S154 and Q215 side chains and that ca. 2 kcal/mol is from a tightening in the stabilizing interactions of the Y217 and R235 side chains. The sum of the effects of individual A154S, A215Q, F217Y and A235R substitutions at the quadruple mutant of ScOMPDC to give the corresponding triple mutants, 5.6 kcal/mol, is much smaller than 16.0 kcal/mol, the sum of the effects of the related four substitutions in wild-type ScOMPDC to give the respective single mutants. The small effect of substitutions at the quadruple mutant is consistent with a large entropic cost to holding the flexible loops of ScOMPDC in the active closed conformation.