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PubMed1984

Mechanisms of enzymatic and acid-catalyzed decarboxylations of prephenate.

The prephenate dehydrogenase activity of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase from Escherichia coli catalyzes the oxidative decarboxylation of both prephenate and deoxoprephenate, which lacks the keto group in the side chain (V 78% and V/K 18% those of prephenate). Hydride transfer is to the B side of NAD, and the acetylpyridine and pyridinecarboxaldehyde analogues of NAD have V/K values 40 and 9% and V values 107 and 13% those of NAD. Since the 13C isotope effect on the decarboxylation is 1.0103 with deuterated and 1.0033 with unlabeled deoxoprephenate (the deuterium isotope effect on V/K is 2.34), the mechanism is concerted, and if CO2 has no reverse commitment, the intrinsic 13C and deuterium isotope effects are 1.0155 (corresponding to a very early transition state for C-C bond cleavage) and 7.3, and the forward commitment is 3.7. With deoxodihydroprephenate (lacking one double bond in the ring), oxidation occurs without decarboxylation, and one enantiomer has a V/K value 23-fold higher than the other (deuterium isotope effects are 3.6 and 4.1 for fast and slow isomers; V for the fast isomer is 5% and V/K 0.7% those of prephenate). The fully saturated analogue of deoxoprephenate is a very slow substrate (V 0.07% and V/K approximately 10(-5%) those of prephenate). pH profiles show a group with pK = 8.3 that must be protonated for substrate binding and a catalytic group with pK = 6.5 that is a cationic acid (likely histidine). This group facilitates hydride transfer by beginning to accept the proton from the 4-hydroxyl group of prephenate prior to the beginning of C-C cleavage (or fully accepting it in the oxidation of the analogues with only one double bond or none in the ring). In contrast with the enzymatic reaction, the acid-catalyzed decarboxylation of prephenate and deoxoprephenate (t1/2 of 3.7 min at low pH) is a stepwise reaction with a carbonium ion intermediate, since 18O is incorporated into substrate and its epi isomer during reaction in H218O. pH profiles show that the hydroxyl group must be protonated and the carboxyl (pK approximately 4.2) ionized for carbonium ion formation. The carbonium ion formed from prephenate decarboxylates 1.75 times faster than it reacts with water (giving 1.8 times as much prephenate as epi isomer). The observed 13C isotope effect of 1.0082 thus corresponds to an intrinsic isotope effect of 1.023, indicating an early transition state for the decarboxylation step. epi-Prephenate is at least 20 times more stable to acid than prephenate because it exists largely as an internal hemiketal.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbon Isotopes↗

PubMed1977

Studies on the biosynthesis of 16-membered macrolide antibiotics using carbon-13 nuclear magnetic resonance spectroscopy.

The origin of the skeletal carbons in the lactone ring of 16-membered macrolide antiobiotics has been studied. 13C-labeled antibiotics leucomycin and tylosin, have been obtained from the culture broth of Streptomyces kitasatoensis 66-14-3 and Streptomyces fradiae C-373, respectively in the presence of appropriate 13C-labeled precursors, and 13C NMR spectra of the antibiotics thus obtained have been measured. It was shown that the aglycone of leucomycin A3 is derived from five acetates, one propionate, one butyrate, and an unknown precursor corresponding to two carbons. The formyl carbon which is characteristic of the basic 16-membered macrolides orginates from C-4 butyrate. On the other hand, the aglycone of tylosin is formed from two acetates, five propionates and one butyrate. Butyric acid and ethylmalonic acid are metabolized to propionyl-CoA or methylmolonyl-CoA through a pathway involving methylmalonyl-CoA mutase, and subsequently incorporated into the lactone ring of tylosin.

Acetates↗

PubMed2000

Probing the catalytic mechanism of prephenate dehydratase by site-directed mutagenesis of the Escherichia coli P-protein dehydratase domain.

The Escherichia coli bifunctional P-protein, which plays a central role in L-phenylalanine (Phe) biosynthesis, contains distinct chorismate mutase (CM) and prephenate dehydratase (PDT) domains as well as a regulatory (R) domain for feedback control by Phe. To elucidate the catalytic mechanism of PDT in the P-protein, 24 mutations of 15 conserved residues in the PDT domain were created, expressed in the pheA(-)E. coli strain NK6024, and studied for their effect on PDT activity. Fourteen mutant enzymes were purified to homogeneity, tested for feedback inhibition by Phe, and characterized by kinetic analysis and circular dichroism spectroscopy. Selected mutant enzymes were further studied by gel filtration, fluorescence emission, and microcalorimetry. In addition, a monofunctional PDT domain (PDT20, residues 101-285) was cloned and overexpressed in plasmid pET with expression levels up to 200-250 mg/L. PDT20 retained full PDT activity, lacked CM activity, and was insensitive to feedback inhibition by Phe. Four residues (T278, N160, Q215, and S208) were shown to be important for PDT catalysis. The values of k(cat)/K(m) for the S208A/C and T278S mutant enzymes were 100-fold lower, and 500-fold lower for the N160A and Q215A mutant enzymes than the wild-type (WT) protein. The T278A and T278V mutant enzymes displayed no measurable catalytic activity, yet bound both prephenate and a competitive inhibitor (S-DNBA) comparably to the WT protein. These data, taken together with the normal CD spectra of the mutant enzymes, strongly suggested that T278 was involved in the catalytic mechanism. To establish whether acidic residues were involved in catalysis, all the conserved Glu and Asp residues in the PDT domain were mutated to Ala. None of these mutations significantly reduced PDT activity, indicating that the acidic residues of the PDT domain are not directly involved in catalysis. However, two mutant enzymes (E159A and E232A) displayed higher levels of PDT activity (2.2- and 3.5-fold, respectively), which was due to enhanced substrate binding. For the double mutant enzyme (E159A-E232A), k(cat)/K(m) was ca. 7-fold higher than for the WT enzyme, while its K(m) was 4.6-fold lower.

Amino Acid Substitution↗

PubMed2006

B12 trafficking in mammals: A for coenzyme escort service.

Many coenzymes are vitamins that are assimilated in mammals into their active form from precursors obtained from the diet. They are often both rare and reactive rendering the likelihood low that the cell uses a collision-based strategy for their delivery to dependent enzymes. In humans, there are only two known B12 or cobalamin-dependent enzymes: methionine synthase and methylmalonyl-CoA mutase. However, the pathway for intracellular assimilation and utilization of this cofactor is complex as revealed by careful clinical analyses of fibroblasts from patients with disorders of cobalamin metabolism. In the recent past, six of the eight human genes involved in the B12 pathway have been identified and these have yielded important insights into their roles. The recent literature on the encoded proteins is reviewed, and a model for intracellular B12 trafficking is proposed in which B12 is escorted to its target proteins in the cytoplasmic and mitochondrial compartments in complex with chaperones, thereby averting problems of dilution and adventitious side reactions.

Animals↗

PubMed2006

DFT analysis of co-alkyl and co-adenosyl vibrational modes in B12-cofactors.

Density functional theory (DFT)-based normal mode calculations have been carried out on models for B12-cofactors to assign reported isotope-edited resonance Raman spectra, which isolate vibrations of the organo-Co group. Interpretation is straightforward for alkyl-Co derivatives, which display prominent Co-C stretching vibrational bands. DFT correctly reproduces Co-C distances and frequencies for the methyl and ethyl derivatives. However, spectra are complex for adenosyl derivatives, due to mixing of Co-C stretching with a ribose deformation coordinate and to activation of modes involving Co-C-C bending and Co-adenosyl torsion. Despite this complexity, the computed spectra provide a satisfactory re-assignment of the experimental data. Reported trends in adenosyl-cobalamin spectra upon binding to the methylmalonyl CoA mutase enzyme, as well as on subsequent binding of substrates and inhibitors, provide support for an activation mechanism involving substrate-induced deformation of the adenosyl ligand.

Cobamides↗

PubMed2001

Interconversion of (S)-glutamate and (2S,3S)-3-methylaspartate: a distinctive B(12)-dependent carbon-skeleton rearrangement.

The interconversion of (S)-glutamate and (2S,3S)-3-methylaspartate catalyzed by B(12)-dependent glutamate mutase is discussed using results from high-level ab initio molecular orbital calculations. Evidence is presented regarding the possible role of coenzyme-B(12) in substrate activation and product formation via radical generation. Calculated electron paramagnetic resonance parameters support experimental evidence for the involvement of substrate-derived radicals and will hopefully aid the future detection of other important radical intermediates. The height of the rearrangement barrier for a fragmentation-recombination pathway, calculated with a model that includes neutral amino and carboxylic acid substituents in the migrating glycyl group, supports recent experimental evidence for the interconversion of (S)-glutamate and (2S,3S)-3-methylaspartate through such a pathway. Our calculations suggest that the enzyme may facilitate the rearrangement of (S)-glutamate through (partial) proton-transfer processes that control the protonation state of substituents in the migrating group.

Aspartic Acid↗

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↗

PubMed2006

Hybrid quantum mechanics/molecular mechanics simulations with two-dimensional interpolated corrections: application to enzymatic processes.

Hybrid quantum mechanics/molecular mechanics (QM/MM) techniques are widely used to study chemical reactions in large systems. Because of the computational cost associated with the high dimensionality of these systems, the quantum description is usually restricted to low-level methods, such as semiempirical Hamiltonians. In some cases, the description obtained at this computational level is quite poor and corrections must be considered. We here propose a simple but efficient way to include higher-level corrections to be used in potential energy surface explorations and in the calculation of potentials of mean force. We evaluate a correction energy term as the difference between a high-level and a low-level calculation on the QM subsystem, employing either the polarized or the gas-phase wave function, obtained as a function of two geometrical coordinates relevant in the process considered. Through the use of two-dimensional bicubic splines this correction energy is included in the simulations, ensuring the continuity and derivability of the energy function. We have tested the proposed scheme with two prototypical examples: the chorismate to prephenate rearrangement catalyzed by Bacillus subtilis chorismate mutase and the catechol methylation catalyzed by catechol O-methyltransferase. In both cases the use of interpolated corrections clearly improves the energetic and geometric descriptions of the reaction.

Bacillus subtilis↗

PubMed2006

Quantitative analysis of mitochondrial protein expression in methylmalonic acidemia by two-dimensional difference gel electrophoresis.

Isolated methylmalonic acidemia (MMA) is a rare metabolic disease due to the deficient activity of L-methylmalonyl-CoA mutase (MCM). This mitochondrial enzyme converts L-methylmalonyl-CoA to succinyl-CoA using adenosylcobalamin (Adocbl) as cofactor. Isolated MMA is subdivided into five forms: mut MMA associated with MCM deficiency, three different defects related to mitochondrial Adocbl formation (cblA, cblB, and cblH), and cblD variant 2. We performed proteomic analysis on mitochondria from an individual with cblH/cblD disorder using 2-D DIGE to identify differentially expressed proteins in this disease. Comparative analysis of control/patient mitochondrial proteome allowed us to identify differential expression of 10 proteins. The most notable groups included proteins involved in apoptosis (cytochrome c), oxidative stress (manganese superoxide dismutase) and cell metabolism (succinyl-CoA ligase (GDP forming) and mitochondrial glycerophosphate dehydrogenase). Immunoblot analysis further validated 2-D DIGE results of two of these proteins in multiple MMA patients, suggesting that the differences in expression are a general effect in this disorder. It is feasible that the differential proteins identified in this study have a biological significance and might be related to the pathophysiology of MMA.

Amino Acid Metabolism, Inborn Errors↗

PubMed1997

Neurological dysfunction in methylmalonic acidaemia is probably related to the inhibitory effect of methylmalonate on brain energy production.

Methylmalonic acidaemia is an inherited metabolic disorder caused by a severe deficiency of the activity of the enzyme L-methylmalonyl-CoA mutase or its cofactor 5'-deoxyadenosylcobalamin, resulting in tissue accumulation of large quantities of methylmalonic acid. Among the various clinical features, neurological symptoms are frequently observed. Patients may present cerebral atrophy and basal ganglia abnormalities are common. In the present report, we update the current knowledge on the influence of methylmalonic acid on brain metabolism in the hope of better understanding the neurological dysfunction characteristic of methylmalonic acidaemia. We present evidence showing that the metabolite inhibits brain energy production by various mechanisms and propose that a fall in cellular ATP generation leading to excitotoxicity is crucial for the occurrence of the neurological damage observed in these patients.

Adenosine Triphosphate↗

PubMed1998

Renal transplantation in a patient with methylmalonic acidaemia.

Renal insufficiency is frequently reported in mutase-deficient methylmalonic acidaemia. We present a case report of a patient with mut- methylmalonic acidaemia who developed chronic tubulointerstitial nephropathy during adolescence. At 24 years of age, she developed end-stage renal failure and underwent renal transplantation. Both plasma and urine methylmalonic acid levels decreased significantly with improved renal function following transplantation. Complications included cyclosporin toxicity and development of diabetes. Renal, metabolic, and clinical status remained improved at 3 years after the kidney transplant.

Adult↗

PubMed2002

Direct NMR observation and DFT calculations of a hydrogen bond at the active site of a 44 kDa enzyme.

A hydrogen bond between the amide backbone of Arg7 and the remote imidazole side chain of HisIO6 has been directly observed by improved TROSY-NMR techniques in the 44 kDa trimeric enzyme chorismate mutase from Bacillus subtilis. The presence of this hydrogen bond in the free enzyme and its complexes with a transition state analog and the reaction product was demonstrated by measurement of 15N-15N and 1H-15N trans-hydrogen bond scalar couplings, (2h)J(NN) and (lh)J(HN), and by transfer of nuclear polarization across the hydrogen bond. The conformational dependences of these coupling constants were analyzed using sum-over-states density functional perturbation theory (SOS-DFPT). The observed hydrogen bond might stabilize the scaffold at the active site of BsCM. Because the Arg7-His 106 hydrogen bond has not been observed in any of the high resolution crystal structures of BsCM, the measured coupling constants provide unique information about the enzyme and its complexes that should prove useful for structural refinement of atomic models.

Arginine↗

PubMed2003

A new strategy for backbone resonance assignment in large proteins using a MQ-HACACO experiment.

A new strategy of backbone resonance assignment is proposed based on a combination of the most sensitive TROSY-type triple resonance experiments such as TROSY-HNCA and TROSY-HNCO with a new 3D multiple-quantum HACACO experiment. The favourable relaxation properties of the multiple-quantum coherences and signal detection using the (13)C' antiphase coherences optimize the performance of the proposed experiment for application to larger proteins. In addition to the (1)H(N), (15)N,(13)C(alpha) and (13)C' chemical shifts the 3D multiple-quantum HACACO experiment provides assignment for the (1)H(alpha) resonances in constrast to previously proposed experiments for large proteins. The strategy is demonstrated with the 44 kDa uniformly (15)N,(13)C-labeled and fractionally 35% deuterated trimeric B. subtilis Chorismate Mutase measured at 20 degrees C and 9 degrees C. Measurements at the lower temperature indicate that the new strategy can be applied to even larger proteins with molecular weights up to 80 kDa.

Bacillus subtilis↗

PubMed2003

A novel strategy for the assignment of side-chain resonances in completely deuterated large proteins using 13C spectroscopy.

The assignment of the aliphatic (13)C resonances of trimeric Bacillus Subtilis chorismate mutase, a protein with a molecular mass of 44 kDa, consisting of three 127-residue monomers is presented by use of two-dimensional (2D) (13)C-start and (13)C-observe NMR experiments. These experiments start with (13)C excitation and end with (13)C observation while relying on the long transverse relaxation times of (13)C spins in uniformly deuterated and (13)C,(15)N-labeled large proteins. Gains in sensitivity are achieved by the use of a paramagnetic relaxation enhancement agent to reduce (13)C T(1) relaxation times with little effect on (13)C T(2) relaxation times. Such 2D (13)C-only NMR experiments circumvent problems associated with the application of conventional experiments for side-chain assignment to proteins of larger sizes, for instance, the absence or low concentration of the side-chain (1)H spins, the transfer of the side-chain spin polarization to the (1)H(N) spins for signal acquisition, or the necessity of a quantitative reprotonation of the methyl moieties in the otherwise fully deuterated side-chains. We demonstrate that having obtained a nearly complete assignment of the side-chain aliphatic (13)C resonances, the side-chain (1)H chemical shifts can be assigned in a semiautomatic fashion using 3D (15)N-resolved and (13)C-resolved NOESY experiments measured with a randomly partially protonated protein sample. We also discuss perspectives for structure determination of larger proteins by using novel strategies which are based on the (1)H,(1)H NOEs in combination with multiple residual dipolar couplings between adjacent (13)C spins determined with 2D (13)C-only experiments.

Amino Acid Sequence↗

PubMed2005

Target discovery in small-molecule cell-based screens by in situ proteome reactivity profiling.

Chemical genomics aims to discover small molecules that affect biological processes through the perturbation of protein function. However, determining the protein targets of bioactive compounds remains a formidable challenge. We address this problem here through the creation of a natural product-inspired small-molecule library bearing protein-reactive elements. Cell-based screening identified a compound, MJE3, that inhibits breast cancer cell proliferation. In situ proteome reactivity profiling revealed that MJE3, but not other library members, covalently labeled the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), resulting in enzyme inhibition. Interestingly, MJE3 labeling and inhibition of PGAM1 were observed exclusively in intact cells. These results support the hypothesis that cancer cells depend on glycolysis for viability and promote PGAM1 as a potential therapeutic target. More generally, the incorporation of protein-reactive compounds into chemical genomics screens offers a means to discover targets of bioactive small molecules in living systems, thereby enabling downstream mechanistic investigations.

Antineoplastic Agents↗

PubMed2001

N219Y, a new frequent mutation among mut(degree) forms of methylmalonic acidemia in Caucasian patients.

Mutations in the MUT locus encoding for the methylmalonyl-CoA mutase (MCM) apoenzyme are responsible for the mut forms of methylmalonic acidemia (MMA). To date, 49 different mutations have been identified in mut MMA. Only two frequent mutations have been reported in the Japanese population and in African-Americans. Here we report a new missense mutation N219Y (731 A-->T) which we found in five unrelated families of French and Turkish descent. All the patients exhibited a severe mut(degree) phenotype and three of them were homozygotes for N219Y. Direct involvement of the mutation in the loss of enzyme activity was demonstrated by mutagenesis and transient expression study. Mapping of the mutation onto a three-dimensional model of human MCM constructed by homology with the Propionibacterium shermanii enzyme shows that it lies in a highly conserved secondary structure motif and might suggest impaired folding and/or poor stability compatible with the mut(degree) phenotype. Finally, a 1% N219Y carrier frequency was observed in a French anonymous control population. Thus, N219Y is the first frequent mut mutation to be reported in the Caucasian population.

Amino Acid Sequence↗

PubMed2003

Theoretical insights in enzyme catalysis.

In this tutorial review we show how the methods and techniques of computational chemistry have been applied to the understanding of the physical basis of the rate enhancement of chemical reactions by enzymes. This is to answer the question: Why is the activation free energy in enzyme catalysed reactions smaller than the activation free energy observed in solution? Two important points of view are presented: Transition State (TS) theories and Michaelis Complex (MC) theories. After reviewing some of the most popular computational methods employed, we analyse two particular enzymatic reactions: the conversion of chorismate to prephenate catalysed by Bacillus subtilis chorismate mutase, and a methyl transfer from S-adenosylmethionine to catecholate catalysed by catechol O-methyltransferase. The results and conclusions obtained by different authors on these two systems, supporting either TS stabilisation or substrate preorganization, are presented and compared. Finally we try to give a unified view, where a preorganized enzyme active site, prepared to stabilise the TS, also favours those reactive conformations geometrically closer to the TS.

Bacillus subtilis↗

PubMed1976

Yeast phosphoglycerate mutate. Cyanogen bromide cleavage and amino acid sequence of an active-site peptide.

The molecular weight and amino acid composition of phosphoglycerate mutase from yeast were determined. CNBr cleavage produced a large (190-residue) fragment and a small (60-residue) fragment. Tryptic and chymotryptic peptides derived from the large fragment were fractionated by ion-exchange chromatography. Peptides from two histidine-containing regions were isolated and the amino acid sequences were determined. Correlation of these data with X-ray-crystallographic evidence shows that the histidine residue in the sequence Arg-Leu Asn-Glu-Arg-His-Tyr-Gly-Asp-Leu-Glu-Gly-Lys is located at the active site.

Amino Acid Sequence↗