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At least 883 records · Page 49Linked to original sources
PubMed2006

Experimental and computational investigation of the uncatalyzed rearrangement and elimination reactions of isochorismate.

The versatile biosynthetic intermediate isochorismate decomposes in aqueous buffer by two competitive pathways, one leading to isoprephenate by a facile Claisen rearrangement and the other to salicylate via elimination of the enolpyruvyl side chain. Computation suggests that both processes are concerted but asynchronous pericyclic reactions, with considerable C-O cleavage in the transition state but relatively little C-C bond formation (rearrangement) or hydrogen atom transfer to the enolpyruvyl side chain (elimination). Kinetic experiments show that rearrangement is roughly 8-times more favorable than elimination. Moreover, transfer of the C2 hydrogen atom to C9 was verified by monitoring the decomposition of [2-(2)H]isochorismate, which was prepared chemoenzymatically from labeled shikimate, by (2)H NMR spectroscopy and observing the appearance of [3-(2)H]pyruvate. Finally, the isotope effects obtained with the C2 deuterated substrate are in good agreement with calculations assuming pericyclic reaction mechanisms. These results provide a benchmark for mechanistic investigations of isochorismate mutase and isochorismate pyruvate lyase, the enzymes that respectively catalyze the rearrangement and elimination reactions in plants and bacteria.

Chorismate Mutase↗
PubMed2003

New quinoproteins in oxidative fermentation.

Several quinoproteins have been newly indicated in acetic acid bacteria, all of which can be applied to fermentative or enzymatic production of useful materials by means of oxidative fermentation. (1) D-Arabitol dehydrogenase from Gluconobacter suboxydans IFO 3257 was purified from the bacterial membrane and found to be a versatile enzyme for oxidation of various substrates to the corresponding oxidation products. It is worthy of notice that the enzyme catalyzes D-gluconate oxidation to 5-keto-D-gluconate, whereas 2-keto-D-gluconate is produced by a flavoprotein D-gluconate dehydrogenase. (2) Membrane-bound cyclic alcohol dehydrogenase was solubilized and purified for the first time from Gluconobacter frateurii CHM 9. When compared with the cytosolic NAD-dependent cyclic alcohol dehydrogenase crystallized from the same strain, the reaction rate in cyclic alcohol oxidation by the membrane enzyme was 100 times stronger than the cytosolic NAD-dependent enzyme. The NAD-dependent enzyme makes no contribution to cyclic alcohol oxidation but contributes to the reduction of cyclic ketones to cyclic alcohols. (3) Meso-erythritol dehydrogenase has been purified from the membrane fraction of G. frateurii CHM 43. The typical properties of quinoproteins were indicated in many respects with the enzyme. It was found that the enzyme, growing cells and also the resting cells of the organism are very effective in producing L-erythrulose. Dihydroxyacetone can be replaced by L-erythrulose for cosmetics for those who are sensitive to dihydroxyacetone. (4) Two different membrane-bound D-sorbitol dehydrogenases were indicated in acetic acid bacteria. One enzyme contributing to L-sorbose production has been identified to be a quinoprotein, while another FAD-containing D-sorbitol dehydrogenase catalyzes D-sorbitol oxidation to D-fructose. D-Fructose production by the oxidative fermentation would be possible by the latter enzyme and it is superior to the well-established D-glucose isomerase, because the oxidative fermentation catalyzes irreversible one-way oxidation of D-sorbitol to D-fructose without any reaction equilibrium, unlike D-glucose isomerase. (5) Quinate dehydrogenase was found in several Gluconobacter strains and other aerobic bacteria like Pseudomonas and Acinetobacter strains. It has become possible to produce dehydroquinate, dehydroshikimate, and shikimate by oxidative fermentation. Quinate dehydrogenase was readily solubilized from the membrane fraction by alkylglucoside in the presence of 0.1 M KCl. A simple purification by hydrophobic chromatography gave a highly purified quinate dehydrogenase that was monodispersed and showed sufficient purity. When quinate dehydrogenase purification was done with Acinetobacter calcoaceticus AC3, which is unable to synthesize PQQ, purified inactive apo-quinate dehydrogenase appeared to be a dimer and it was converted to the monomeric active holo-quinate dehydrogenase by the addition of PQQ.

Alcohol Oxidoreductases↗
PubMed1990

sym 13-A Gene Conditioning Ineffective Nodulation in Pisum sativum.

Treatment of Pisum sativum (L.) cv. ;Sparkle' with ethyl methanesulfonic acid (EMS) produced a stable mutant, E135F, which forms small, white, ineffective nodules. These nodules exhibit histological zonation typical of an indeterminant nodule, e.g. meristematic, early symbiotic, late symbiotic, and senescent zones. Compared with the nitrogen fixing nodules of the parent, the zones are smaller and the nodules senesce prematurely. Bacteroids in E135F are less elongated and less differentiated than those in ;Sparkle.' The E135F mutant forms ineffective nodules when inoculated with nine different effective strains of Rhizobium leguminosarum and also when grown in a soil containing effective strains. The ineffective phenotype of E135F is under monogenic recessive control; the gene is designated sym 13. sym 13 was located on chromosome 2 by linkage with genes for shikimic dehydrogenase and esterase-2. The original selection E135F carried another mutation in heterozygous form at a separate locus, yielding some homozygous recessive nonnodulating progeny, E135N, in later generations. This indicates that EMS treatments may cause mutations at more than one sym gene. The gene conditioning non-nodulation in E135N was designated sym 14. It mapped to a locus on a different part of chromosome 2 by linkage to the gene for fumarase. The data demonstrate that sym genes are not necessarily closely linked.

Journal Article↗
PubMed1992

Inactivation of 5-enolpyruvylshikimate 3-phosphate synthase by its substrate analogue pyruvate in the presence of sodium cyanoborohydride.

Incubation of Escherichia coli 5-enolpyruvylshikimate 3-phosphate synthase with its substrate analogue pyruvate in the presence of sodium cyanoborohydride resulted in a significant decrease in its enzyme activity. The inactivation followed pseudo-first order and saturation kinetics with a Kinact of 16.12 mM and a maximum rate of constant of 0.046 min-1. The inactivation was specifically prevented by preincubation of the enzyme with a combination of the substrate shikimate 3-phosphate plus competitive inhibitor glyphosate. Upon 90% inactivation, approximately 1 mole of [14C]-label from [14C]-pyruvate was incorporated per mole of enzyme. Tryptic mapping of the modified enzyme as well as analysis of an isolated radioactive peptide indicated that Lys22 was the modified site. The results suggested that pyruvate inactivated the enzyme by forming a Schiff-base with Lys22 at the enzyme's active site.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMedDate not supplied

Tyrosine biosynthesis in Sorghum bicolor: isolation and regulatory properties of arogenate dehydrogenase.

The conversion of prephenic acid to tyrosine can occur by two different routes: (a) oxidative decarboxylation (prephenate dehydrogenase) followed by transamination (aromatic aminotransferase); (b) transamination of prephenate forming the non-aromatic amino acid arogenic acid (prephenate aminotransferase) followed by oxidative decarboxylation (arogenate dehydrogenase). High activity of arogenate dehydrogenase was found in extracts of etiolated sorghum seedlings, while no evidence of prephenate dehydrogenase was observed. Arogenate dehydrogenase from sorghum eluted, with high recovery of activity (93%), as a single peak on DEAE-cellulose chromatography. The enzyme was strongly inhibited by tyrosine but was unaffected by phenylalanine, prephenate, or tryptophan. Kinetic analysis showed that tyrosine inhibition was competitive with arogenate and that the Ki for tyrosine (61 microM) was much smaller than the Km for arogenate (350 microM). The properties of arogenate dehydrogenase indicate that this enzyme is important in the regulation of tyrosine biosynthesis in sorghum. Strong inhibition of the enzyme by tyrosine may indicate that arogenate is a branch point in the shikimate pathway in plants and therefore arogenate may be a precursor to phenylalanine and the numerous phenylpropanoid secondary metabolites derived from phenylalanine.

Chromatography, DEAE-Cellulose↗
PubMed2006

Biochemical characterization and inhibitor discovery of shikimate dehydrogenase from Helicobacter pylori.

Shikimate dehydrogenase (SDH) is the fourth enzyme involved in the shikimate pathway. It catalyzes the NADPH-dependent reduction of 3-dehydroshikimate to shikimate, and has been developed as a promising target for the discovery of antimicrobial agent. In this report, we identified a new aroE gene encoding SDH from Helicobacter pylori strain SS1. The recombinant H. pylori shikimate dehydrogenase (HpSDH) was cloned, expressed, and purified in Escherichia coli system. The enzymatic characterization of HpSDH demonstrates its activity with k(cat) of 7.7 s(-1) and K(m) of 0.148 mm toward shikimate, k(cat) of 7.1 s(-1) and K(m) of 0.182 mm toward NADP, k(cat) of 5.2 s(-1) and K(m) of 2.9 mm toward NAD. The optimum pH of the enzyme activity is between 8.0 and 9.0, and the optimum temperature is around 60 degrees C. Using high throughput screening against our laboratory chemical library, five compounds, curcumin (1), 3-(2-naphthyloxy)-4-oxo-2-(trifluoromethyl)-4H-chromen-7-yl 3-chlorobenzoate (2), butyl 2-{[3-(2-naphthyloxy)-4-oxo-2-(trifluoromethyl)-4H-chromen-7-yl]oxy}propanoate (3), 2-({2-[(2-{[2-(2,3-dimethylanilino)-2-oxoethyl]sulfanyl}-1,3-benzothiazol-6-yl)amino]-2-oxoethyl}sulfanyl)-N-(2-naphthyl)acetamide (4), and maesaquinone diacetate (5) were discovered as HpSDH inhibitors with IC(50) values of 15.4, 3.9, 13.4, 2.9, and 3.5 microm, respectively. Further investigation indicates that compounds 1, 2, 3, and 5 demonstrate noncompetitive inhibition pattern, and compound 4 displays competitive inhibition pattern with respect to shikimate. Compounds 1, 4, and 5 display noncompetitive inhibition mode, and compounds 2 and 3 show competitive inhibition mode with respect to NADP. Antibacterial assays demonstrate that compounds 1, 2, and 5 can inhibit the growth of H. pylori with MIC of 16, 16, and 32 microg.mL(-1), respectively. This current work is expected to favor better understanding the features of SDH and provide useful information for the development of novel antibiotics to treat H. pylori-associated infection.

Alcohol Oxidoreductases↗
PubMed1999

Site-directed mutagenesis of putative active site residues of 5-enolpyruvylshikimate-3-phosphate synthase.

The site-directed mutagenesis of a number of proposed active site residues of 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase is reported. Several of these mutations resulted in complete loss of enzyme activity indicating that these residues are probably involved with catalysis, notably K22R, K411R, D384A, R27A, R100A, and D242A. Of those, K22R, R27A, and D384A did not bind either the substrate shikimate-3-phosphate (S3P) or glyphosate (GLP). The K411R and D242A mutants bind S3P only in the presence of GLP. The kinetic characterization of mutants R100K, K340R, and E418A, which retain activity, is reported. Of those, R100K and K340R do not accumulate enzyme intermediate of enzyme-bound product under equilibrium conditions. These residues, while not essential for catalysis, are most likely important for substrate binding. All of the mutants are shown to be correctly folded by NMR spectroscopy.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1996

The transient kinetics of Escherichia coli chorismate synthase: substrate consumption, product formation, phosphate dissociation, and characterization of a flavin intermediate.

Chorismate synthase is the seventh enzyme of the shikimate pathway and catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. The reaction involves the 1,4-elimination of phosphate and the C-(6proR) hydrogen of the substrate with unusual anti stereochemistry and requires a reduced flavin cofactor. This paper describes the kinetics of the formation and decay of a flavin intermediate, EPSP consumption, chorismate and phosphate formation, and phosphate dissociation during single and multiple turnover experiments, determined using rapid reaction techniques. The kinetics of phosphate dissociation using the substrate analogues (6R)-[6-2H]EPSP and (6S)-6-fluoro-EPSP have also been determined. The observations are consistent with a nonconcerted chorismate synthase reaction. The flavin intermediate is not simply associated with the conversion of substrate to product because it forms before the substrate is consumed. The transient spectral changes must be associated primarily with events such as protonation of the reduced flavin, a charge transfer complex between reduced flavin and an aromatic amino acid, or a conformational change in the protein. This does not rule out the direct role of flavin in catalysis.

Escherichia coli↗
PubMed2004

Site-directed mutagenesis of the active site region in the quinate/shikimate 5-dehydrogenase YdiB of Escherichia coli.

YdiB and its paralog AroE are members of the quinate/shikimate 5-dehdrogenase family. Enzymes from this family function in the shikimate pathway that is essential for survival of microorganisms and plants and represent potential drug targets. Recent YdiB and AroE crystal structures revealed the presence of a NAD(P)-binding and a catalytic domain. We carried out site-directed mutagenesis of 8 putative active site residues in YdiB from Escherichia coli and analyzed structural and kinetic properties of the mutant enzymes. Our data indicate critical roles for an invariant lysine and aspartate residue in substrate binding and allowed us to differentiate between two previously proposed models for the binding of the substrate in the active site. Comparison of several YdiB and AroE structures led us to conclude that, upon cofactor binding and domain closure, the 2 identified binding residues are repositioned to bind to the substrate. Although the lysine residue contributes to some extent to the stabilization of the transition state, we did not identify any residue as catalytically essential. This indicates that catalysis does not operate through a general acid-base mechanism, as thought originally. Our improved understanding of the medically and agriculturally important quinate/shikimate 5-dehydrogenase family at the molecular level may prove useful in the development of novel herbicides and antimicrobial agents.

Alcohol Oxidoreductases↗
PubMed2004

Solute transporters as connecting elements between cytosol and plastid stroma.

Metabolite translocators in the inner membrane of the plastid envelope are the interface between cytosolic and plastidial metabolism. Hence, they integrate plastidial pathways, such as photosynthesis, starch biosynthesis, the oxidative pentose phosphate pathway and the shikimate pathway, into the metabolic network of plant cells. Metabolite transporters not only catalyze the flux of metabolites between compartments but also represent information pathways that communicate the metabolic status of the various compartments within plant cells. Recently, a pentose-phosphate translocator was shown to be a novel member of the phosphate translocator protein family. Furthermore, a protein of previously unknown function was identified as a novel type of maltose transporter, and a glutamate/malate translocator that is involved in photorespiration was discovered. In addition, the pathway for maltose metabolism in the cytosol has been unraveled.

Arabidopsis↗
PubMed2003

Engineered biosynthesis of an ansamycin polyketide precursor in Escherichia coli.

Ansamycins such as rifamycin, ansamitocin, and geldanamycin are an important class of polyketide natural products. Their biosynthetic pathways are especially complex because they involve the formation of 3-amino-5-hydroxybenzoic acid (AHBA) followed by backbone assembly by a hybrid nonribosomal peptide synthetase/polyketide synthase. We have reconstituted the ability to synthesize 2,6-dimethyl-3,5,7-trihydroxy-7-(3'-amino-5'-hydroxyphenyl)-2,4-heptadienoic acid (P8/1-OG), an intermediate in rifamycin biosynthesis, in an extensively manipulated strain of Escherichia coli. The parent strain, BAP1, contains the sfp phosphopantetheinyl transferase gene from Bacillus subtilis, which posttranslationally modifies polyketide synthase and nonribosomal peptide synthetase modules. AHBA biosynthesis in this host required introduction of seven genes from Amycolatopsis mediterranei, which produces rifamycin, and Actinosynnema pretiosum, which produces ansamitocin. Because the four-module RifA protein (530 kDa) from the rifamycin synthetase could not be efficiently produced in an intact form in E. coli, it was genetically split into two bimodular proteins separated by matched linker pairs to facilitate efficient inter-polypeptide transfer of a biosynthetic intermediate. A derivative of BAP1 was engineered that harbors the AHBA biosynthetic operon, the bicistronic RifA construct and the pccB and accA1 genes from Streptomyces coelicolor, which enable methylmalonyl-CoA biosynthesis. Fermentation of this strain of E. coli yielded P8/1-OG, an N-acetyl P8/1-OG analog, and AHBA. In addition to providing a fundamentally new route to shikimate and ansamycin-type compounds, this result enables further genetic manipulation of AHBA-derived polyketide natural products with unprecedented power.

Actinomycetales↗
PubMed2001

Subcellular localization and characterization of chorismate synthase in the apicomplexan Plasmodium falciparum.

The resurgence of drug-resistant apicomplexa, in particular Plasmodium falciparum, the most fatal human malarial parasite, has focused attention on the recent discovery of the shikimate pathway in these organisms, as it may provide the urgently required, novel drug targets resulting from the absence of this pathway in mammals. The direction of a parasiticidal drug design programme obviously requires knowledge of the subcellular localization and indeed full characterization of the possible enzyme targets. Here, we report the cloning and characterization of chorismate synthase from P. falciparum and present the first biochemical and immunological studies of an enzyme of the shikimate pathway from an apicomplexan parasite. We show that this chorismate synthase does not possess an intrinsic flavin reductase activity and is therefore monofunctional like the plant and bacterial chorismate synthases. Highest immunological cross-reactivity was found with a plant chorismate synthase. However, in contrast to the plant enzyme, which is located to the plastid, P. falciparum chorismate synthase is found in the parasite cytosol, akin to the fungal enzymes that possess an intrinsic flavin reductase activity (i.e. are bifunctional). Thus, P. falciparum chorismate synthase has a combination of properties that distinguishes it from other described chorismate synthases.

Amino Acid Sequence↗
PubMed2006

A structural model for chorismate synthase from Mycobacterium tuberculosis in complex with coenzyme and substrate.

The enzymes of the shikimate pathway constitute an excellent target for the design of new antibacterial agents; chorismate synthase (CS) catalyzes the last step of this pathway. The prediction of Mycobacterium tuberculosis (MTB) CS three-dimensional structure and the geometric docking of the coenzyme FMN and the substrate EPSP were performed using the crystal structure of CS from Streptococcus pneumoniae as template. Energy minimization of the whole complex showed, as expected, that most of the template interactions are preserved in the MTB structure, except for HIS11, ARG139 and GLN255. However, novel interactions involving ARG111, GLY113 and SER317 were also observed.

Amino Acid Sequence↗
PubMed1993

Genesis of eukaryotic transcriptional activator and repressor proteins by splitting a multidomain anabolic enzyme.

The genes necessary for the correctly regulated catabolism of quinate in Aspergillus nidulans and Neurospora crassa are controlled at the level of transcription by a DNA-binding activator protein and a repressor protein that directly interact with one another. The repressor protein is homologous throughout its length with the three C-terminal domains of a pentafunctional enzyme catalysing five consecutive steps in the related anabolic shikimate pathway. We now report that the activator protein is homologous to the two N-terminal domains of the same pentafunctional enzyme and that this proposed structural similarity suggests a molecular mechanism by which the repressor recognises the activator protein. We believe that this is the first report of the genesis of a pair of interacting eukaryotic regulatory proteins by the splitting of a multidomain anabolic enzyme. The recruitment of preformed enzymatically active domains to a regulatory role may represent a general mechanism for the evolution of pathway-specific regulator proteins in dispensable pathways.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1995

Background-minimized cassette mutagenesis by PCR using cassette-specific selection markers: a useful general approach for studying structure-function relationships of multisubstrate enzymes.

An efficient protocol, termed background-minimized cassette mutagenesis (BMCM) by PCR, has been developed for multiple mutagenesis of DNA. This method uses suitable extension primers for incorporating various mutation(s) and is not limited by either the nature of the mutation or the size and spatial location of mutational loci. Minimization of the wild type background clone and mutant selection at very high frequency were easily achieved through a two-step process. First, a deletion of a unique restriction site within the cassette was introduced through additional silent mutation(s). Then, the recombinant clones were digested with the corresponding enzyme followed by transformation when selective linearization of wild-type clone led to its near total removal leaving the mutant clones as the only practicable transformants. Because it is generally possible to design several such cassette-specific unique background minimization markers for any gene, for multiple mutagenesis involving distally located portions of the gene the present protocol is superior to other currently available methods. The efficiency of BMCM-PCR has been demonstrated here by using the multisubstrate enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPs) of Bacillus subtilis as a model system. Three different sets of cassettes of varying sizes were generated to encompass the three putative active/binding regions in the beginning, middle, and the end of the gene encoding EPSPs. Very high efficiency of mutation incorporation and selection were obtained in all cases. Furthermore, by taking advantage of the unique cassette-specific background elimination markers, it was possible to generate a nested set of double and/or triple mutants. The mutant enzymes were overexpressed in Escherichia coli and purified to near homogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2003

Evolution of feedback-inhibited beta /alpha barrel isoenzymes by gene duplication and a single mutation.

The betaalpha barrel is the common protein fold of numerous enzymes and was proposed recently to be the result of gene duplication and fusion of an ancient half-barrel. The initial enzyme of shikimate biosynthesis possesses the additional feature of feedback regulation. The crystal structure and kinetic studies on chimera and mutant proteins of yeast 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase from Saccharomyces cerevisiae inhibited by phenylalanine (Aro3p) and DAHP synthase S. cerevisiae inhibited by tyrosine (Aro4p) give insight into important regions for regulation in the enzyme: The loop, which is connecting the two half-barrels, and structural elements added to the barrel are prerequisites for regulation and form a cavity on the N-terminal side of the betaalpha barrel. In the cavity of Aro4p at position 226 is a glycine residue, which is highly conserved in all other tyrosine-regulated DAHP synthases as well. Sequence alignments with phenylalanine-regulated DAHP synthases including Aro3p show a highly conserved serine residue at this position. An exchange of glycine to serine and vice versa leads to a complete change in the regulation pattern. Therefore the evolution of these differently feedback-inhibited isoenzymes required gene duplication and a single mutation within the internal extra element. Numerous additional amino acid substitutions present in the contemporary isoenzymes are irrelevant for regulation and occurred independently.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed2002

Crystal structure of shikimate kinase from Mycobacterium tuberculosis reveals the dynamic role of the LID domain in catalysis.

Shikimate kinase (SK) and other enzymes in the shikimate pathway are potential targets for developing non-toxic antimicrobial agents, herbicides, and anti-parasite drugs, because the pathway is essential in the above species but is absent from mammals. The crystal structure of Mycobacterium tuberculosis SK (MtSK) in complex with MgADP has been determined at 1.8 A resolution, revealing critical information for the structure-based design of novel anti-M. tuberculosis agents. MtSK, with a five-stranded parallel beta-sheet flanked by eight alpha-helices, has three domains: the CORE domain, the shikimate-binding domain (SB), and the LID domain. The ADP molecule is bound with its adenine moiety sandwiched between the side-chains of Arg110 and Pro155, its beta-phosphate group in the P-loop, and the alpha and beta-phosphate groups hydrogen bonded to the guanidinium group of Arg117. Arg117 is located in the LID domain, is strictly conserved in SK sequences, is observed for the first time to interact with any bound nucleotide, and appears to be important in both substrate binding and catalysis. The crystal structure of MtSK (this work) and that of Erwinia chrysanthemi SK suggest a concerted conformational change of the LID and SB domains upon nucleotide binding.

Adenosine Diphosphate↗
PubMed1994

Site-directed mutagenesis and NMR studies of histidine-385 mutants of 5-enolpyruvylshikimate-3-phosphate synthase.

The site-directed mutagenesis of His-385 of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is reported. The steady-state kinetics for two mutants, H385Q and H385A, are compared with that of the wild-type enzyme. H385Q EPSP synthase was found to have 25% wild-type enzyme activity, whereas H385A EPSP synthase retained 1% activity. The KM values for Pi and shikimate 3-phosphate were unaffected, whereas the KM for phosphoenolpyruvate (PEP) was increased 10 times for H385Q EPSP synthase. The KM for EPSP was unaffected in H385Q but raised by a factor of 10 in H385A EPSP synthase. The binding of glyphosate was studied by fluorescence spectroscopy and by 31P NMR spectroscopy. Direct observation of the enzyme-intermediate complexes by 13C NMR spectroscopy with [2,3-13C]phosphoenolpyruvate was studied for the mutant enzymes and compared with the wild type. Under equilibrium conditions, H385A EPSP synthase does not accumulate enzyme-bound EPSP. These results suggest that, while critically located in the PEP binding site, His-385 is not the residue responsible for initiating catalysis through the protonation of PEP.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗