Assessing the economic value of antihypertensive medications.

OBJECTIVE: To assess the economic value of antihypertensive medications by comparing the likelihood of coronary heart disease and stroke events and subsequent event treatment costs. STUDY DESIGN: Duration of blood pressure reduction was used to profile event risk reduction of three antihypertensive medications. METHODS: We used clinical data to determine the duration of blood pressure reduction achieved with use of two angiotensin converting enzyme inhibitors and one angiotensin II receptor antagonist. We then used trough-to-peak ratios to calculate the reduction in risk of coronary heart disease and stroke events associated with each medication. RESULTS: Across a number of different event treatment cost and population size estimates, the economic value of different medications can be assessed. CONCLUSION: Our method for assessing the economic value of antihypertensive medications can be applied to other drug classes and can be further refined by integrating patient population and other risk-related data.

A lysine to arginine substitution at position 146 of rabbit aldolase A changes the rate-determining step to Schiff base formation.

Lys146 of rabbit aldolase A [D-fructose-1,6-bis(phosphate): D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13] was changed to arginine by site-directed mutagenesis. The kcat of the resulting mutant protein, K146R, was 500 times slower than wild-type in steady-state kinetic assays for both cleavage and condensation of fructose-1,6-bis(phosphate), while the K(m) for this substrate was unchanged. Analysis of the rate of formation of catalytic intermediates showed K146R was significantly different from the wild-type enzyme and other enzymes mutated at this site. Single-turnover experiments using acid precipitation to trap the Schiff base intermediate on the wild-type enzyme failed to show a build-up of this intermediate on K146R. However, K146R retained the ability to form the Schiff base intermediate as shown by the significant amounts of Schiff base intermediate trapped with NaBH4. In the single-turnover experiments it appeared that the Schiff base intermediate was converted to products more rapidly than it was produced. This suggested a maximal rate of Schiff base formation of 0.022 s-1, which was close to the value of kcat for this enzyme. This observation is strikingly different from the wild-type enzyme in which Schiff base formation is > 100 times faster than kcat. For K146R it appears that steps up to and including Schiff base formation are rate limiting for the catalytic reaction. The carbanion intermediate derived from either substrate or product, and the equilibrium concentrations of covalent enzyme-substrate intermediates, were much lower on K146R than on the wild-type enzyme. The greater bulk of the guanidino moiety may destabilize the covalent enzyme-substrate intermediates, thereby slowing the rate of Schiff base formation such that it becomes rate limiting. The K146R mutant enzyme is significantly more active than other enzymes mutated at this site, perhaps because it maintains a positively charged group at an essential position in the active site or perhaps the Arg functionally substitutes as a general acid/base catalyst in both Schiff base formation and in subsequent abstraction of the C4-hydroxyl proton.

Preliminary analysis of crystals of 4-oxalocrotonate tautomerase, an enzyme composed of unusually small monomers.

Crystals of 4-oxalocrotonate tautomerase have been obtained by the vapor-diffusion method using polyethylene glycol as precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a = 118.1, b = 95.6, c = 97.4, alpha = beta = gamma = 90 degrees and diffract well to at least 2.7 A resolution. There are approximately ten 6811 dalton subunits of the enzyme per asymmetric unit, giving a crystal solvent content of 70%.

Comparison of the refined crystal structures of liganded and unliganded chicken, yeast and trypanosomal triosephosphate isomerase.

The refined crystal structures of chicken, yeast and trypanosomal triosephosphate isomerase (TIM) have been compared. TIM is known to exist in an "open" (unliganded) and "closed" (liganded) conformation. For chicken TIM only the refined open structure is available, whereas for yeast TIM and trypanosomal TIM refined structures of both the open and the closed structure have been used for this study. Comparison of these structures shows that the open structures of chicken TIM, yeast TIM and trypanosomal TIM are essentially identical. Also it is shown that the closed structures of yeast TIM and trypanosomal TIM are essentially identical. The conformational difference between the open and closed structures concerns a major shift (7 A) in loop-6. Minor shifts are observed in the two adjacent loops, loop-5 (1 A) and loop-7 (1 A). The pairwise comparison of the three different TIM barrels shows that the 105C alpha atoms of the core superimpose within 0.9 A. The sequences of these three TIMs have a pairwise sequence identity of approximately 50%. The residues that line the active site are 100% conserved. The residues interacting with each other across the dimer interface show extensive variability, but the direct hydrogen bonds between the two subunits are well conserved. The orientation of the two monomers with respect to each other is almost identical in the three different TIM structures. There are 56 (22%) conserved residues out of approximately 250 residues in 13 sequences. The functions of most of these conserved residues can be understood from the available open and closed structures of the three different TIMs. Some of these residues are quite far from the active site. For example, at a distance of 19 A from the active site there is a conserved saltbridge interaction between residues at the C-terminal ends of alpha-helix-6 and alpha-helix-7. This anchoring contrasts with the large conformational flexibility of loop-6 and loop-7 near the N termini of these helices. The flexibility of loop-6 is facilitated by a conserved large empty cavity near the N terminus of alpha-helix-6, which exists only in the open conformation.

Structure of the triosephosphate isomerase-phosphoglycolohydroxamate complex: an analogue of the intermediate on the reaction pathway.

The glycolytic enzyme triosephosphate isomerase (TIM) catalyzes the interconversion of the three-carbon sugars dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (GAP) at a rate limited by the diffusion of substrate to the enzyme. We have solved the three-dimensional structure of TIM complexed with a reactive intermediate analogue, phosphoglycolohydroxamate (PGH), at 1.9-A resolution and have refined the structure to an R-factor of 18%. Analysis of the refined structure reveals the geometry of the active-site residues and the interactions they make with the inhibitor and, by analogy, the substrates. The structure is consistent with an acid-base mechanism in which the carboxylate of Glu-165 abstracts a proton from carbon while His-95 donates a proton to oxygen to form an enediol (or enediolate) intermediate. The conformation of the bound substrate stereoelectronically favors proton transfer from substrate carbon to the syn orbital of Glu-165. The crystal structure suggests that His-95 is neutral rather than cationic in the ground state and therefore would have to function as an imidazole acid instead of the usual imidazolium. Lys-12 is oriented so as to polarize the substrate oxygens by hydrogen bonding and/or electrostatic interaction, providing stabilization for the charged transition state. Asn-10 may play a similar role.

Computer simulation and analysis of the reaction pathway of triosephosphate isomerase.

A theoretical approach designed for chemical reactions in the condensed phase is used to determine the energy along the reaction path of the enzyme triosephosphate isomerase. The calculations address the role of the enzyme in lowering the barrier to reaction and provide a decomposition into specific residue contributions. The results suggest that, although Lys-12 is most important, many other residues within 16 A of the substrate contribute and that histidine-95 as the imidazole/imidazolate pair could act as an acid/base catalyst.

Structure of yeast triosephosphate isomerase at 1.9-A resolution.

The structure of yeast triosephosphate isomerase (TIM) has been solved at 3.0-A resolution and refined at 1.9-A resolution to an R factor of 21.0%. The final model consists of all non-hydrogen atoms in the polypeptide chain and 119 water molecules, a number of which are found in the interior of the protein. The structure of the active site clearly indicates that the carboxylate of the catalytic base, Glu 165, is involved in a hydrogen-bonding interaction with the hydroxyl of Ser 96. In addition, the interactions of the other active site residues, Lys 12 and His 95, are also discussed. For the first time in any TIM structure, the "flexible loop" has well-defined density; the conformation of the loop in this structure is stabilized by a crystal contact. Analysis of the subunit interface of this dimeric enzyme hints at the source of the specificity of one subunit for another and allows us to estimate an association constant of 10(14)-10(16) M-1 for the two monomers. The analysis also suggests that the interface may be a particularly good target for drug design. The conserved positions (20%) among sequences from 13 sources ranging on the evolutionary scale from Escherichia coli to humans reveal the intense pressure to maintain the active site structure.

Triosephosphate isomerase: removal of a putatively electrophilic histidine residue results in a subtle change in catalytic mechanism.

An important active-site residue in the glycolytic enzyme triosephosphate isomerase is His-95, which appears to act as an electrophilic component in catalyzing the enolization of the substrates. With the techniques of site-directed mutagenesis, His-95 has been replaced by Gln in the isomerase from Saccharomyces cerevisiae. The mutant isomerase has been expressed in Escherichia coli strain DF502 and purified to homogeneity. The specific catalytic activity of the mutant enzyme is less than that of wild type by a factor of nearly 400. The mutant enzyme can be resolved from the wild-type isomerase on nondenaturing isoelectric focusing gels, and an isomerase activity stain shows that the observed catalytic activity indeed derives from the mutant protein. The inhibition constants for arsenate and for glycerol phosphate with the mutant enzyme are similar to those with the wild-type isomerase, but the substrate analogues 2-phosphoglycolate and phosphoglycolohydroxamate bind 8- and 35-fold, respectively, more weakly to the mutant isomerase. The mutant enzyme shows the same stereospecificity of proton transfer as the wild type. Tritium exchange experiments similar to those used to define the free energy profile for the wild-type yeast isomerase, together with a new method of analysis involving 14C and 3H doubly labeled substrates, have been used to investigate the energetics of the mutant enzyme catalyzed reaction. When the enzymatic reaction is conducted in tritiated solvent, the mutant isomerase does not catalyze any appreciable exchange between protons of the remaining substrate and those of the solvent either in the forward reaction direction (using dihydroxyacetone phosphate as substrate) or in the reverse direction (using glyceraldehyde phosphate as substrate). However, the specific radioactivity of the product glyceraldehyde phosphate formed in the forward reaction is 31% that of the solvent, while that of the product dihydroxyacetone phosphate formed in the reverse reaction is 24% that of the solvent. The deuterium kinetic isotope effects observed with the mutant isomerase using [1(R)-2H]dihydroxyacetone phosphate and [2-2H]glyceraldehyde 3-phosphate are 2.15 +/- 0.04 and 2.4 +/- 0.1, respectively. These results lead to the conclusion that substitution of Gln for His-95 so impairs the ability of the enzyme to stabilize the reaction intermediate that there is a change in the pathways of proton transfer mediated by the mutant enzyme. The data allow us more closely to define the role of His-95 in the reaction catalyzed by the wild-type enzyme, while forcing us to be alert to subtle changes in mechanistic pathways when mutant enzymes are generated.

Subunit interface of triosephosphate isomerase: site-directed mutagenesis and characterization of the altered enzyme.

We have replaced asparagine residues at the subunit interface of yeast triosephosphate isomerase (TIM) using site-directed mutagenesis in order to elucidate the effects of substitutions on the catalytic activity and conformational stability of the enzyme. The mutant proteins were expressed in a strain of Escherichia coli lacking the bacterial isomerase and purified by ion-exchange and immunoadsorption chromatography. Single replacements of Asn-78 by either Thr or Ile residues had little effect on the enzyme's catalytic efficiency, while the single replacement Asn-78----Asp-78 and the double replacement Asn-14/Asn-78----Thr-14/Ile-78 appreciably lowered kcat for the substrate D-glyceraldehyde 3-phosphate. The isoelectric point of the mutant Asn-78----Asp-78 was equivalent to that of wild-type yeast TIM that had undergone a single, heat-induced deamidation, and this mutant enzyme was less resistant than wild-type TIM to denaturation and inactivation caused by elevated temperature, denaturants, tetrabutylammonium bromide, alkaline pH, and proteases.