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K S Anderson

Publications and source records attributed to K S Anderson.

At least 55 records · Page 3Linked to original sources
PubMed1996

Intersubunit communication in tryptophan synthase by carbon-13 and fluorine-19 REDOR NMR.

The beta subunits of the 143-kDa alpha2beta2 tetrameric enzyme tryptophan synthase have been labeled by L-[ring-4-19F]phenylalanine and L-[phenol-4-13C]tyrosine in an effort to monitor the positions of these residues on ligand binding. Of the 13 phenylalanine and 11 tyrosine residues in the beta subunit, only three pairs have labels with 13C-19F separations of less than 6 angstrom. The beta subunit residues Tyr279 and Phe280 (each members of one of the three Tyr-Phe proximate pairs) have been suggested as possible conformational gates on ligand binding. The 188-MHz 19F NMR spectrum of the microcrystalline, double-labeled enzyme complex has five resolved lines under 5-kHz magic-angle spinning and 80-kHz proton dipolar decoupling. The distribution of beta-subunit 19F isotropic shifts is altered by addition of L-[3-13C]-serine to the mother liquor in contact with the microcrystals, consistent with a conformational rearrangement. The 13C label from serine is detected at 28 ppm as a methyl tautomer of bound aminoacrylate. The change in aromatic 19F chemical shifts on binding of serine indicates an alteration in local electric field gradients within the beta subunit. However, rotational-echo double-resonance 13C NMR (with 19F dephasing) shows that the average 13C-19F distance for the three phenylalanine-tyrosine proximate pairs in the beta subunit is changed by less than 1 angstrom.

Carbon Isotopes↗
PubMed1996

HIV-1 reverse transcriptase resistance to nonnucleoside inhibitors.

The parameters governing the polymerization mechanism of reverse transcriptase containing the tyrosine to cysteine mutation at position 181 (Y181C) were determined using pre-steady-state techniques. The pathway for single nucleotide incorporation catalyzed by Y181C is similar to that determined for wild-type RT where a rate-limiting conformational change precedes fast chemistry and is followed by slow steady-state release of the primer/template. The Y181C mutant enzyme binds a 25/45-mer duplex DNA tightly with a Kd of 11 nM. However, the Y181C mutation weakens the nucleotide affinity 2-3-fold relative to the wild-type complex. We also determined the parameters governing the mechanism of nonnucleoside inhibitor resistance with Y181C. The Kd value of Nevirapine with the mutant E.DNA complex increased approximately 500-fold. The decreased affinity of Nevirapine for the mutant enzyme is a consequence of a faster inhibitor dissociation rate from the enzyme complex of Y181C relative to that of the wild-type. The E.DNA complex of Y181C may be saturated with Nevirapine, and the I.E.DNA complex is capable of a maximum incorporation rate of 0.1 s-1 (a 10-fold faster rate than that of the wild-type I.E.DNA complex). The overall two-step binding of nucleotide to Y181C in the presence of Nevirapine remains unaffected.

Antiviral Agents↗
PubMed1996

Surface point mutations that significantly alter the structure and stability of a protein's denatured state.

Significantly different m values (1.9-2.7 kcal mol-1 M-1) were observed for point mutations at a single, solvent-exposed site (T53) in a variant of the B1 domain of streptococcal Protein G using guanidine hydrochloride (GuHCl) as a denaturant. This report focuses on elucidating the energetic and structural implications of these m-value differences in two Protein G mutants, containing Ala and Thr at position 53. These two proteins are representative of the high (m+) and low (m-) m-value mutants studied. Differential scanning calorimetry revealed no evidence of equilibrium intermediates. A comparison of GuHCl denaturation monitored by fluorescence and circular dichroism showed that secondary and tertiary structure denatured concomitantly. The rates of folding (286 S-1 for the m+ mutant and 952 S-1 for the m- mutant) and the rates of unfolding (11 S-1 for m+ mutant and 3 S-1 for the m- mutant) were significantly different, as determined by stopped-flow fluorescence. The relative solvation free energies of the transition states were identical for the two proteins (alpha ++ = 0.3). Small-angle X-ray scattering showed that the radius of gyration of the denatured state (Rgd) of the m+ mutant did not change with increasing denaturant concentrations (Rgd approximately 23 A); whereas, the Rgd of the m- mutant increased from approximately 17 A to 23 A with increasing denaturant concentration. The results indicate that the mutations exert significant effects in both the native and GuHCl-induced denatured state of these two proteins.

Bacterial Proteins↗
PubMed1996

Ocular abnormalities in congenital cytomegalovirus infection.

BACKGROUND: Congenital cytomegalovirus (CMV) infection often results in damage to the brain, auditory system and visual system that leads to debility. This report describes the frequency, severity and effect on vision function of ocular abnormalities in a large cohort of children with congenital CMV infection. METHODS: Serial eye examinations were performed on 445 infants and children with congenital CMV infection from one month to 9 years of age, assessing anatomic integrity, motor sensory function, retina and posterior pole. RESULTS: Ocular disease was manifest principally by chorioretinitis, optic atrophy, pigmentary retinopathy and strabismus; each of these findings occurred more frequently (p < 0.05) among children who were symptomatic at birth than among those who were initially asymptomatic. These same findings were also more common among patients born after a primary maternal infection than among those born after a recurrent maternal infection. More than half of the patients with symptomatic congenital CMV infection with chorioretinitis or optic atrophy had significant binocular vision impairment. CONCLUSIONS: Ocular disease is an important sequela of congenital CMV infection that can lead to impaired vision. In order to allow for early intervention, patients with congenital CMV infection should have eye examinations beginning in early infancy.

Alabama↗
PubMed1995

Kinetic characterization of channel impaired mutants of tryptophan synthase.

Tryptophan synthase, an alpha 2 beta 2 tetrameric complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the alpha subunit to the active site of the beta subunit. The solution of the three-dimensional structure of the enzyme from Salmonella typhimurium provided physical evidence for a 25-A hydrophobic tunnel which connects the alpha and beta active sites (Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871). Using rapid reaction kinetics, we have previously established that indole is indeed channeled and have identified three essential kinetic features which govern efficient channeling. In the current study we have probed the necessity of these features by using site-directed mutagenesis to alter these requirements. We now report the kinetic characterization of two mutants which contain substitutions to block or restrict the tunnel (beta C170F and beta C170W). Preliminary kinetic and structural evidence of a restricted tunnel in the beta C170W has been provided (Schlichting, I., Yang, X. W., Miles, E. W., Kim, A. Y., and Anderson, K. S. (1994) J. Biol. Chem. 269, 26591-26593). The rapid kinetic analysis of these mutant proteins shows that these mutations interfere with efficient channeling of the indole metabolite such that indole can be observed in single enzyme turnover of the physiologically relevant alpha beta reaction. In addition, the beta C170W mutant appears to be impaired in alpha beta intersubunit communication.

Binding Sites↗
PubMed1995

Reevaluating glyphosate as a transition-state inhibitor of EPSP synthase: identification of an EPSP synthase.EPSP.glyphosate ternary complex.

Numerous studies have confirmed that glyphosate forms a tight ternary complex with EPSP synthase and shikimate 3-phosphate. It has been proposed [Anton, D., Hedstrom, L., Fish, S., & Abeles, R. (1983) Biochemistry 22, 5903-5908; Steinrücken, H. C., & Amrhein, N. (1984) Eur. J. Biochem. 143, 351-357] that in this complex glyphosate functions as a transition-state analog of the putative phosphoenolpyruvoyl oxonium ion. For this to be true, glyphosate must occupy the space in the enzyme active site that is normally associated with PEP and, through turnover, the carboxyvinyl group of the product EPSP. According to this model, one would predict that, in the reverse EPSP synthase reaction with EPSP and phosphate as substrates, there should be little if any interaction of glyphosate with enzyme or enzyme.substrate complexes. In contrast to this expectation, rapid gel filtration experiments provided direct evidence that glyphosate could be trapped on the enzyme in the presence of EPSP to form a ternary complex of EPSPS.EPSP.glyphosate. The experimentally determined stoichiometry for this complex, 0.62 equiv of glyphosate/mole of EPSPS, is similar to that found for the EPSPS.S3P.glyphosate ternary complex (0.66). This direct binding result was corroborated and quantitated by fluorescence titration experiments which demonstrated that glyphosate forms a reasonably tight (Kd = 56 +/- 1 microM) ternary complex with enzyme and EPSP. This finding was further verified, and its impact on substrate turnover analyzed, by steady-state kinetics. Glyphosate was found to be an uncompetitive inhibitor versus EPSP with Kii(app) = 54 +/- 2 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1995

Expression of human cyclophilin-40 and the effect of the His141-->Trp mutation on catalysis and cyclosporin A binding.

The cDNA encoding a human cytosolic 40-kDa cyclophilin (CyP-40) has been inserted into a modified pGEX-3X expression vector and expressed in Escherichia coli to yield recombinant CyP-40 at levels up to 4 mg/l medium. The protein was purified to homogeneity using a cyclosporin affinity matrix and gel filtration. The recombinant CyP-40 showed peptidyl-prolyl cis-trans isomerase activity (kcat/Km = 1.12 x 10(6) M-1.s-1) comparable to that of bovine brain CyP-40. The weak affinity of CyP-40 for cyclosporin A was postulated to arise from a histidine residue that replaces a tryptophan residue critical for cyclosporin A binding and highly conserved in other cyclophilins that have high affinity for cyclosporin A. Site-directed mutagenesis to replace His141 by tryptophan yielded a protein with an approximately 20-fold greater affinity for cyclosporin A (Kdapp 11.5 +/- 2 nM as determined by tryptophan fluorescence measurements). The intrinsic isomerase activity of this mutant protein with succinyl-Ala-Ala-Pro-Phe 4-nitroanilide as substrate was about nine times greater than the value obtained for the nonmutated recombinant CyP-40 and had an activity similar to that of CyP-18. NMR difference spectroscopy and molecular modelling revealed a cyclosporin-A-binding domain that is similar to that of CyP-18.

Amino Acid Isomerases↗
PubMed1995

Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleoside inhibitors.

The mechanism of inhibition of HIV-1 reverse transcriptase by three nonnucleoside inhibitors is described. Nevirapine, O-TIBO, and CI-TIBO each bind to a hydrophobic pocket in the enzyme-DNA complex close to the active site catalytic residues. Pre-steady-state kinetic analysis was used to establish the mechanism of inhibition by these noncompetitive inhibitors. Analysis of the pre-steady-state burst of DNA polymerization indicated that inhibitors blocked the chemical reaction, but did not interfere with nucleotide binding or the nucleotide-induced conformational change. Rather, in the presence of saturating concentrations of the inhibitors, the nucleoside triphosphate bound tightly (Kd, 100 nM), but nonproductively. The data suggest that an inhibitor combining the functionalities of a nonnucleoside inhibitor and a nucleotide analog could bind very tightly and specifically to reverse transcriptase and could be effective in the treatment of AIDS.

Antiviral Agents↗
PubMed1994

Structural and kinetic analysis of a channel-impaired mutant of tryptophan synthase.

Substrate channeling is a process by which two sequential enzymes interact to transfer a metabolite (or intermediate) from one enzyme active site to the next without allowing free diffusion of the metabolite. Channeling is thought to play an important role in metabolic regulation and cellular modulation of enzymatic activities. Although there are numerous examples of sequential enzyme pairs in glycolysis and other biosynthetic pathways that are thought to exhibit channeling, this is as yet controversial. Tryptophan synthase is considered the best example for an enzyme displaying channeling behavior between subunits. Tryptophan synthase is an alpha 2 beta 2 tetrameric enzyme complex, which catalyzes the last two steps in the biosynthesis of L-tryptophan. The alpha subunit catalyzes the cleavage of indole-3-glycerol phosphate to indole and glyceraldehyde-3-phosphate; the beta subunit catalyzes the condensation of indole with serine to form tryptophan, in a reaction mediated by pyridoxal phosphate. The inability to trap free indole in the steady-state reaction and analysis of the kinetics of single turnover reactions have led to the postulate that indole may pass directly from the alpha to the beta site without diffusion through solution (Demoss, J. A. (1962) Biochim. Biophys. Acta 62, 279-293; Matchett, W. M. (1974) J. Biol. Chem. 249, 4041-4049). The crystal structure of tryptophan synthase from Salmonella typhimurium has provided additional support for substrate channeling by elucidating a 25-A hydrophobic tunnel connecting the two catalytic sites (Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871). The structure suggests that mutation of a residue lining the tunnel to a more bulky residue might impede or block the passage of indole during catalysis thus enabling detection of indole during a single enzyme turnover. A mutant of tryptophan synthase has been prepared in which one of the residues lining the tunnel, beta Cys-170, has been replaced with a bulkier tryptophan residue. Kinetic and structural analyses of the beta C170W mutant by rapid chemical quench methods and x-ray crystallographic analysis show both the transient formation of indole and the obstruction of the tunnel, thus providing direct evidence for the substrate channeling mechanism.

Catalysis↗
PubMed1994

Detection and characterization of a phospholactoyl-enzyme adduct in the reaction catalyzed by UDP-N-acetylglucosamine enolpyruvoyl transferase, MurZ.

The MurZ enzyme catalyzes enolpyruvoyl transfer from phosphoenolpyruvate to the 3-OH of UDP-N-acetylglucosamine (UDP-GlcNAc) in Escherichia coli peptidoglycan biosynthesis. The kinetic mechanism of MurZ has been shown to involve the generation of a non-covalently bound tetrahedral phospholactoyl-UDP-GlcNAc intermediate [Marquardt, J.L., et al. (1993) J. Am. Chem. Soc. 115, 10398-10399]. In the work described here, MurZ overproduced in E. coli copurified with 1 equiv of bound PEP. Enzyme free of bound PEP was prepared by incubation of purified MurZ with UDP-GlcNAc followed by removal of small molecules. Addition of either [14C]PEP or [32P]PEP to PEP-free enzyme led to stoichiometric labeling. The MurZ-PEP complex was stable to 6.7 M urea, and digestion of the [32P]PEP-labeled protein followed by SDS-PAGE generated a labeled peptide of molecular weight 5000. Solution NMR of MurZ incubated with [2-13C]PEP suggested a tetrahedral phospholactoyl enzyme adduct attached via C-2 to an enzyme nucleophile. The kon for generation of the phospholactoyl enzyme in the absence of UDP-GlcNAc was 0.24 microM-1 s-1, too slow to represent the binding order of the kinetically preferred pathway since the lower limit of the second-order binding constant for PEP (kcat/Km) is 15 microM-1 s-1. Rapid chemical quench analysis under single-turnover conditions using [32P]PEP in the presence of UDP-GlcNAc demonstrated that the covalent enzyme-phospholactoyl adduct appeared and decayed on a time scale consistent with catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkyl and Aryl Transferases↗
PubMed1994

A role for calnexin (IP90) in the assembly of class II MHC molecules.

Major histocompatibility complex (MHC) class II antigens consist of alpha and beta chains that associate intracellularly with the invariant (I) chain. The HLA-DR alpha beta I complex assembles in the endoplasmic reticulum (ER) into a nonameric structure via progressive addition of three alpha beta dimers to a core invariant chain trimer. We have examined intracellular association of alpha beta I complexes with the resident ER protein calnexin. Calnexin associates rapidly (within 3 min) with newly synthesized alpha, beta and I chains, and remains associated with the assembling alpha beta I complex until the final alpha beta dimer is added, forming the complete nonamer. Dissociation of calnexin parallels egress of alpha beta I from the ER. These results suggest that calnexin retains and stabilizes both free class II subunits and partially assembled class II-I chain complexes until assembly of the nonamer is complete.

Calcium-Binding Proteins↗
PubMed1993

Evidence that transporters associated with antigen processing translocate a major histocompatibility complex class I-binding peptide into the endoplasmic reticulum in an ATP-dependent manner.

We have investigated the role of the putative peptide transporters associated with antigen processing (TAP) by using a permeabilized-cell system. The main objective was to determine whether these molecules, which bear homology to the ATP-binding cassette family of transporters, translocate antigenic peptides across the endoplasmic reticulum membrane for assembly with major histocompatibility complex (MHC) class I molecules and beta 2-microglobulin light chain. The pore-forming toxin streptolysin O was used to generate permeabilized cells, and peptide translocation was determined by measuring the amount of added radiolabeled peptide bound to endogenous class I molecules. No radiolabeled peptide was associated with MHC class I glycoproteins from unpermeabilized cells. We found that efficient peptide binding to MHC class I molecules in permeabilized cells is both transporter dependent and ATP dependent. In antigen-processing mutant cells lacking a functional transporter, uptake occurs only through a less-efficient transporter and ATP-independent pathway. In addition, short peptides (8-10 amino acids) known to bind MHC class I molecules compete efficiently with a radiolabeled peptide for TAP-dependent translocation, whereas longer peptides and a peptide derived from an endoplasmic reticulum signal sequence do not compete efficiently. This result indicates that the optimal substrates for TAP possess the characteristics of MHC-binding peptides.

Adenosine Triphosphate↗
PubMed1993

Intracellular transport of class I MHC molecules in antigen processing mutant cell lines.

Intracellular transport and stability of class I MHC glycoproteins depends on the assembly of H chain, beta 2-microglobulin, and peptide. The Ag processing mutant cell lines T2 and RMA-S have defects in peptide loading of class I, resulting in reduced cell surface expression of class I molecules. Expression of class I molecules in the murine cell line RMA-S can be induced at 26 degrees C, suggesting that they are transported to the cell surface, but are unstable. However, most human class I molecules in T2 are poorly expressed at the cell surface, even at 26 degrees C. To directly compare the transport of human and mouse alleles in RMA-S and T2, the human alleles HLA-A2, A3, and B27 were transfected into RMA-S along with human beta 2-microglobulin, and the mouse alleles H-2Kb and Db were transfected into T2. Surface expression of HLA-A3 and B27 in RMA-S remained less than 10% of wild-type levels at 26 degrees C. H-2Kb and Db in both cell lines, however, were expressed at 20 to 30% wild-type levels at 37 degrees C and could be induced to wild-type levels at 26 degrees C or with peptides. The selective expression of murine class I glycoproteins at the cell surface of T2 is not because of their greater stability when associated with human beta 2m, since H-2Kb and Db H chain/human beta 2m complexes dissociate more rapidly in vitro than HLA-A3 and B27 complexes. These results suggest that the difference in transport between human and mouse class I in T2 reflects a fundamental structural property of the class I glycoproteins.

Alleles↗
PubMed1992

Mechanism and fidelity of HIV reverse transcriptase.

We have examined the RNA-dependent and DNA-dependent polymerase and ribonuclease H catalytic activities of human immunodeficiency virus reverse transcriptase using rapid transient kinetic methods with defined synthetic 25/45-mer DNA/RNA and DNA/DNA primer/templates. The Kd value for interaction of the enzyme with duplex DNA was 4.7 nM, and the value for RNA/DNA heteroduplex was of similar magnitude. A pre-steady state burst of nucleoside triphosphate incorporation was observed for both DNA and RNA templates. Analysis of the dATP concentration dependence of the burst rate provided Kd values for dATP of 4 and 14 microM and maximum rates of single nucleotide incorporation, kpol, of 33 and 74 s-1, for DNA and RNA templates, respectively. Subsequent turnovers were limited by the rate of dissociation of the primer/template from the enzyme at rates of 0.18 and 0.06 s-1 for duplex DNA and RNA/DNA heteroduplex, respectively. Analysis of rates of DNA polymerization and RNA cleavage using the RNA template revealed that the two activities are independent of one another. The polymerization rate (4-70 s-1) was dependent on dATP concentration, whereas the RNA cleavage occurred at a constant rate of 10 s-1 over the 100-fold dATP concentration range (2-200 microM). Examination of the RNA cleavage products resulting from a single turnover indicates that the polymerase and ribonuclease domains of the enzyme are separated by a distance corresponding to 19 bases of RNA/DNA heteroduplex, consistent with the recently published crystal structure (Kohlstaedt, L. A., Wang, J., Friedman, J., Rice, P. A., and Steitz, T. A. (1992) Science 256, 1783-1790). Analysis of the kinetics of processive synthesis suggested that the initial binding of dNTP leads to a faster rate of dissociation of DNA from the enzyme. Further investigation supported a two-step dNTP binding mechanism with the formation of an initial E.DNA.dNTP complex followed by a more stable E'.DNA.dNTP complex. The Kd values for incorporation of incorrect nucleoside triphosphates opposite a DNA template thymidine were 1010 microM for dGTP, 1240 microM for dCTP, and 840 microM for dTTP. The corresponding maximum kpol rates were 4.8 s-1 for dGTP, 0.52 s-1 for dCTP, and 0.41 s-1 for dTTP. These values provide fidelity estimates of 1740 for discrimination against dGTP, 19,700 for dCTP, and 16,900 for dTTP misincorporations at this site.

Base Sequence↗
PubMed1991

Structure and topological symmetry of the glyphosate target 5-enolpyruvylshikimate-3-phosphate synthase: a distinctive protein fold.

5-enol-Pyruvylshikimate-3-phosphate synthase (EPSP synthase; phosphoenolpyruvate:3-phosphoshikimate 1-carboxyvinyltransferase, EC 2.5.1.19) is an enzyme on the pathway toward the synthesis of aromatic amino acids in plants, fungi, and bacteria and is the target of the broad-spectrum herbicide glyphosate. The three-dimensional structure of the enzyme from Escherichia coli has been determined by crystallographic techniques. The polypeptide backbone chain was traced by examination of an electron density map calculated at 3-A resolution. The two-domain structure has a distinctive fold and appears to be formed by 6-fold replication of a protein folding unit comprising two parallel helices and a four-stranded sheet. Each domain is formed from three of these units, which are related by an approximate threefold symmetry axis; in each domain three of the helices are completely buried by a surface formed from the three beta-sheets and solvent-accessible faces of the other three helices. The domains are related by an approximate dyad, but in the present crystals the molecule does not display pseudo-symmetry related to the symmetry of point group 32 because its approximate threefold axes are almost normal. A possible relation between the three-dimensional structure of the protein and the linear sequence of its gene will be described. The topological threefold symmetry and orientation of each of the two observed globular domains may direct the binding of substrates and inhibitors by a helix macrodipole effect and implies that the active site is located near the interdomain crossover segments. The structure also suggests a rationale for the glyphosate tolerance conferred by sequence alterations.

Journal Article↗
PubMed1991

Serine modulates substrate channeling in tryptophan synthase. A novel intersubunit triggering mechanism.

Tryptophan synthase, an alpha 2 beta 2 complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the alpha subunit to the active site of the beta subunit. We now examine the kinetics of substrate channeling by tryptophan synthase directly by chemical quench-flow and stopped-flow methods. The conversion of indole-3-glycerol phosphate (IGP) to tryptophan at the active site proceeds at a rate of 24 s-1, which is limited by the rate of cleavage of IGP to produce indole (alpha reaction). In a single turnover experiment monitoring the conversion of radiolabeled IGP to tryptophan, only a trace of indole is detectable (less than or equal to 1% of the IGP), implying that the reaction of indole to form tryptophan must be quite fast (greater than or equal to 1000 s-1). The rate of reaction of indole from solution is much too slow (40 s-1 under identical conditions) to account for the negligible accumulation of indole in a single turnover. Therefore, the indole produced at the alpha site must be rapidly channeled to the beta site, where it reacts with serine to form tryptophan: channeling and the reaction of indole to form tryptophan must each occur at rates greater than or equal to 1000 s-1. Steady-state turnover is limited by the slow rate of tryptophan release (8 s-1). In the absence of serine, the cleavage of IGP to indole is limited by a change in protein conformation at a rate of 0.16 s-1. When the alpha beta reaction is initiated by mixing enzyme with IGP and serine simultaneously, there is a lag in the cleavage IGP and formation of tryptophan. The kinetics of the lag correspond to the rate of formation of the aminoacrylate in the reaction of serine with pyridoxal phosphate at the beta site, measured by stopped-flow methods (45 s-1). There is also a change in protein fluorescence, suggestive of a change in protein conformation, occurring at the same rate. Substitution of cysteine for serine leads to a longer lag in the kinetics of IGP cleavage and a correspondingly slower rate of formation of the aminoacrylate (6 s-1). Thus, the reaction of serine at the beta site modulates the alpha reaction such that the formation of the aminoacrylate leads to a change in protein conformation that is transmitted to the alpha site to enhance the rate of IGP cleavage 150-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1990

"Kinetic competence" of the 5-enolpyruvoylshikimate-3-phosphate synthase tetrahedral intermediate.

The tetrahedral intermediate formed at the active site of 5-enolpyruvoylshikimate-3-phosphate synthase by reaction of shikimate 3-phosphate with phosphoenolpyruvate was isolated, and its properties in solution and in reaction with enzyme were examined. The intermediate was moderately stable at pH 7.0, with a half-life of 45 min, and showed increasing lifetimes with increasing pH (t1/2 greater than 48 h at pH greater than or equal to 12). The intermediate bound to the enzyme rapidly, with a second order rate constant of 5 x 10(7) M-1 s-1. Upon binding to the enzyme, it reacted to form both products (5-enolpyruvoylshikimate 3-phosphate, Pi) and substrates (shikimate 3-phosphate, phosphoenolpyruvate) in proportions predicted by the rate constants defined previously for reactions occurring at the active enzyme site (Anderson, K.S. Sikorski, J.A., and Johnson, K. A. (1988b) Biochemistry 27, 7395-7406). The kinetics of binding and dissociation of stable phosphonate analogs of the tetrahedral intermediate (Alberg, D., and Bartlett, P.A. (1989) J. Am. Chem. Soc. 111, 2337) were also examined. In comparison to the intermediate, the analogs bound to the enzyme 300-10,000 fold more slowly and at least 300-20,000 times mroe weakly. These results clarify the definitions for kinetic competence of enzyme intermediates and call into question the significance of the slow binding of analogs of transition states or enzyme intermediates.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗