Use of Enrichment Culture for Directed Evolution of the Vibrio fluvialis JS17 ω-Transaminase, Which Is Resistant to Product Inhibition by Aliphatic Ketones

A novel high-throughput screening method that overcame product inhibition was used to isolate a mutant ω-transaminase from Vibrio fluvialis JS17. An enzyme library was generated using error-prone PCR mutagenesis and then enriched on minimal medium containing 2-aminoheptane as the sole nitrogen source and 2-butanone as an inhibitory ketone. An identified mutant enzyme, ω-TAmla, showed significantly reduced product inhibition by aliphatic ketone. The product inhibition constants of the mutant with 2-butanone and 2-heptanone were 6- and 4.5-fold higher than those of the wild type, respectively. Using ω-TAmla (50 U/ml) overexpressed in Escherichia coli BL21, 150 mM 2-aminoheptane was successfully resolved to (R)-2-aminoheptane (enantiomeric excess, >99%) with 53% conversion with an enantioselectivity of >100.     

Trypanosoma brucei: chemical evidence that cathepsin L is essential for survival and a relevant drug target

The protozoan parasite causing human African trypanosomiasis, Trypanosoma brucei, displays cysteine peptidase activity, the chemical inhibition of which is lethal to the parasite. This activity comprises a cathepsin B (TbCATB) and a cathepsin L (TbCATL). Previous RNA interference (RNAi) data suggest that TbCATB rather than TbCATL is essential to survival even though silencing of the latter was incomplete. Also, chemical evidence supporting the essentiality of either enzyme which would facilitate a target-based drug development programme is lacking. Using specific peptidyl inhibitors and substrates, we quantified the contributions of TbCATB and TbCATL to the survival of T. brucei. At 100 μM, the minimal inhibitory concentration that kills all parasites in culture, the non-specific cathepsin inhibitors, benzyloxycarbonyl-phenylalanyl-arginyl-diazomethyl ketone (Z-FA-diazomethyl ketone) and (l-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l-proline methyl ester (CA-074Me) inhibited TbCATL and TbCATB by >99%. The cathepsin L (CATL)-specific inhibitor, ((2S,3S)-oxirane-2,3-dicarboxylic acid 2-[((S)-1-benzylcarbamoyl-2-phenyl-ethyl)-amide] 3-{[2-(4-hydroxy-phenyl)-ethyl]-amide}) (CAA0225), killed parasites with >99% inhibition of TbCATL but only 70% inhibition of TbCATB. Conversely, the cathepsin B (CATB)-specific inhibitor, (l-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l-proline (CA-074), did not affect survival even though TbCATB inhibition at >95% was statistically indistinguishable from the complete inhibition by Z-FA-diazomethyl ketone and CA-074Me. The observed inhibition of TbCATL by CA-074 and CA-074Me was shown to be facilitated by the reducing intracellular environment. All inhibitors, except the CATB-specific inhibitor, CA-074, blockaded lysosomal hydrolysis prior to death. The results suggest that TbCATL, rather than TbCATB, is essential to the survival of T. brucei and an appropriate drug target.     

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.     

Inactivation of the water-oxidizing enzyme in manganese stabilizing protein-free mutant cells of the cyanobacteria Synechococcus PCC7942 and Synechocystic PCC6803 during dark incubation and conditions leading to photoactivation

The previously constructed MSP (manganese stabilizing protein-psbO gene product)-free mutant of Synechococcus PCC7942 (Bockholt R, Masepohl B and Pistorius E K (1991) FEBS Lett 294: 59–63) and a newly constructed MSP-free mutant of Synechocystis PCC6803 were investigated with respect to the inactivation of the water-oxidizing enzyme during dark incubation. O₂ evolution in the MSP-free mutant cells, when measured with a sequence of short saturating light flashes, was practically zero after an extended dark adaptation, while O₂ evolution in the corresponding wild type cells remained nearly constant. It could be shown that this inactivation could be reversed by photoactivation. With isolated thylakoid membranes from the MSP-free mutant of PCC7942, it could be demonstrated that photoactivation required illumination in the presence of Mn²⁺ and Ca²⁺, while Cl^– addition was not required under our experimental conditions. Moreover, an extended analysis of the kinetic properties of the water-oxidizing enzyme (kinetics of the S₃(S₄)S₀ transition, S-state distribution, deactivation kinetics) in wild type and mutant cells of Synechococcus PCC7942 and Synechocystis PCC6803 was performed, and the events possibly leading to the reversible inactivation of the water-oxidizing enzyme in the mutant cells are discussed. We could also show that the water-oxidizing enzyme in the MSP-free mutant cells is more sensitive to inhibition by added NH₄Cl-suggesting that NH₃ might be a physiological inhibitor of the water oxidizing enzyme in the absence of MSP.Abbreviations Chl chlorophyll - DCBQ 2,6-Dichloro-p-benzoquinone - MSP manganese stabilizing protein (psbO gene product) - PS II Photosystem II - WOE water oxidizing enzyme - WT wild type This paper is dedicated to Prof. Dr. Bernard Axelrod on the occasion of his 80th birthday     

Asymmetric reduction of alpha, beta-unsaturated ketone to (R) allylic alcohol by Candida chilensis

A pilot scale whole cell process was developed for the enantioselective 1,2-reduction of prochiral alpha,beta-unsaturated ketone to (R) allylic alcohol using Candida chilensis. Initial development showed high enantiomeric excess (EE > 95%) but low product yield (10%). Process development, using a combination of statistically designed screening and optimization experiments, improved the desired alcohol yield to 90%. The fermentation growth stage, particularly medium composition and growth pH, had a significant impact on the bioconversion while process characterization identified diverse challenges including the presence of multiple enzymes, substrate/product toxicity, and biphasic cellular morphology. Manipulating the fermentation media allowed control of the whole cell morphology to a predominantly unicellular broth, away from the viscous pseudohyphae, which were detrimental to the bioconversion. The activity of a competing enzyme, which produced the undesired saturated ketone and (R) saturated alcohol, was minimized to < or =5% by controlling the reaction pH, temperature, substrate concentration, and biomass level. Despite the toxicity effects limiting the volumetric productivity, a reproducible and scaleable process was demonstrated at pilot scale with high enantioselectivity (EE > 95%) and overall yield greater than 80%. This was the preferred route compared to a partially purified process using ultra centrifugation, which led to improved volumetric productivity but reduced yield (g/day). The whole cell approach proved to be a valuable alternative to chemical reduction routes, as an intermediate step for the asymmetric synthesis of an integrin receptor antagonist for the inhibition of bone resorption and treatment of osteoporosis.     

Kinetic resolution of chiral amines with omega-transaminase using an enzyme-membrane reactor

A kinetic resolution process for the production of chiral amines was developed using an enzyme-membrane reactor (EMR) and a hollow-fiber membrane contactor with (S)-specific omega-transaminases (omega-TA) from Vibrio fluvialis JS17 and Bacillus thuringiensis JS64. The substrate solution containing racemic amine and pyruvate was recirculated through the EMR and inhibitory ketone product was selectively extracted by the membrane contactor until enantiomeric excess of (R)-amine exceeded 95%. Using the reactor set-up with flat membrane reactor (10-mL working volume), kinetic resolutions of alpha-methylbenzylamine (alpha-MBA) and 1-aminotetralin (200 mM, 50 mL) were carried out. During the operation, concentration of ketone product, i.e., acetophenone or alpha-tetralone, in a substrate reservoir was maintained below 0.1 mM, suggesting efficient removal of the inhibitory ketone by the membrane contactor. After 47 and 32.5 h of operation using 5 U/mL of enzyme, 98.0 and 95.5% ee of (R)-alpha-MBA and (R)-1-aminotetralin were obtained at 49.5 and 48.8% of conversion, respectively. A hollow-fiber membrane reactor (39-mL working volume) was used for a preparative-scale kinetic resolution of 1-aminotetralin (200 mM, 1 L). After 133 h of operation, enantiomeric excess reached 95.6% and 14.3 g of (R)-1-aminotetralin was recovered (97.4% of yield). Mathematical modeling of the EMR process including the membrane contactor was performed to evaluate the effect of residence time. The simulation results suggest that residence time should be short to maintain the concentration of the ketone product in EMR sufficiently low so as to decrease conversion per cycle and, in turn, reduce the inhibition of the omega-TA activity.     

Lysine 356 is a critical residue for binding the C-6 phospho group of fructose 2,6-bisphosphate to the fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Lysine 356 has been implicated by protein modification studies as a fructose-2,6-bisphosphate binding site residue in the 6-phosphofructo-2-kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Kitajima, S., Thomas, H., and Uyeda, K. (1985) J. Biol. Chem. 260, 13995-14002). However, Lys-356 is found in the fructose-2,6-bisphosphatase domain (Bazan, F., Fletterick, R., and Pilkis, S. J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). In order to ascertain whether Lys-356 is involved in fructose-2,6-bisphosphatase catalysis and/or domain/domain interactions of the bifunctional enzyme, Lys-356 was mutated to Ala, expressed in Escherichia coli, and then purified to homogeneity. Circular dichroism experiments indicated that the secondary structure of the Lys-356-Ala mutant was not significantly different from that of the wild-type enzyme. The Km for fructose 2,6-bisphosphate and the Ki for the noncompetitive inhibitor, fructose 6-phosphate, for the fructose-2,6-bisphosphatase of the Lys-356-Ala mutant were 2700- and 2200-fold higher, respectively, than those of the wild-type enzyme. However, the maximal velocity and the Ki for the competitive product inhibitor, inorganic phosphate, were unchanged compared to the corresponding values of the wild-type enzyme. Furthermore, in contrast to the wild-type enzyme, which exhibits substrate inhibition, there was no inhibition by substrate of the Lys-356-Ala mutant. In the presence of saturating substrate, inorganic phosphate, which acts by relieving fructose-6-phosphate and substrate inhibition, is an activator of the bisphosphatase. The Ka for inorganic phosphate of the Lys-356-Ala mutant was 1300-fold higher than that of the wild-type enzyme. The kinetic properties of the 6-phosphofructo-2-kinase of the Lys-356-Ala mutant were essentially identical with that of the wild-type enzyme. The results demonstrate that: 1) Lys-356 is a critical residue in fructose-2,6-bisphosphatase for binding the 6-phospho group of fructose 6-phosphate/fructose 2,6-bisphosphate; 2) the fructose 6-phosphate binding site is responsible for substrate inhibition; 3) Inorganic phosphate activates fructose-2,6-bisphosphatase by competing with fructose 6-phosphate for the same site; and 4) Lys-356 is not involved in 6-phosphofructo-2-kinase substrate/product binding or catalysis.     

Potent anti-tumor effects of an active site mutant of human manganese-superoxide dismutase. Evolutionary conservation of product inhibition

Mn-SOD serves as the primary cellular defense against oxidative damage by converting superoxide radicals (O(2)(-)) to O(2) and H(2)O(2). A unique characteristic of this mitochondrial anti-oxidant enzyme is the conservation from bacteria to man of a rapidly formed product inhibited state. Using site-directed mutagenesis, we have generated an active site mutant (H30N) of human Mn-SOD, which exhibits significantly reduced product inhibition and increased enzymatic efficiency. Overexpression of the H30N enzyme causes anti-proliferative effects in vitro and anti-tumor effects in vivo. Our results provide a teleological basis for the phylogenetically invariant nature of position His-30 and the evolutionary conservation of product inhibition. These data also provide more direct intracellular evidence for the signaling role associated with H(2)O(2).     

Simultaneous synthesis of enantiomerically pure (S)-amino acids and (R)-amines using α/ω-aminotransferase coupling reactions with two-liquid phase reaction system

An efficient simultaneous synthesis of enantiopure (S)-amino acids and chiral (R)-amines was achieved using α/ω-aminotransferase (α/ω-AT) coupling reaction with two-liquid phase system. As, among the enzyme components in the α/ω-AT coupling reaction systems, only ω-AT is severely hampered by product inhibition by ketone product, the coupled reaction cannot be carried out above 60 mM substrates. To overcome this problem, a two-liquid phase reaction was chosen, where dioctylphthalate was selected as the solvent based upon biocompatibility, partition coefficient and effect on enzyme activity. Using 100 mM of substrates, the AroAT/ω-AT and the AlaAT/ω-AT coupling reactions asymmetrically synthesized (S)-phenylalanine and (S)-2-aminobutyrate with 93% (>99% ee^S) and 95% (>99% ee^S) of conversion yield, and resolved the racemic α-methylbenzylamine with 56% (95% ee^R) and 54% (96% ee^R) of conversion yield, respectively. Moreover, using 300 mM of 2-oxobutyrate and 300 mM of racemic α-methylbenzylamine as substrates, the coupling reactions yielded 276 mM of (S)-2-aminobutyrate (>99% ee) and 144 mM of (R)-α-methylbenzylamine (>96% ee) in 9 h. Here, most of the reactions take place in the aqueous phase, and acetophenone mainly moved to the organic phase according to its partition coefficient.     

alpha-Bromo ketone substrate analogues are powerful reversible inhibitors of carboxypeptidase A

The aldehyde (RS)-2-benzyl-4-oxobutanoic acid, which is 25% hydrated at pH 7.5, has recently been shown to be a strong reversible competitive inhibitor of carboxypeptidase A [Ki = 0.48 nM; Galardy, R. E., & Kortylewicz, Z. P. (1984) Biochemistry 23, 2083-2087]. The ketone analogue of this aldehyde (RS)-2-benzyl-4-oxopentanoic acid (IV) is not detectably hydrated under the same conditions and is 1500-fold less potent (Ki = 730 microM). The ketone homologue (RS)-2-benzyl-5-oxohexanoic acid (XIII) is also a weak inhibitor (Ki = 1.3 mM). The alpha-monobrominated derivatives of these two ketones are, however, strong competitive inhibitors with Ki's of 0.57 microM and 1.3 microM, respectively. Oximes derived from the aldehyde, the ketones IV and XIII, and a homologue of the aldehyde are weak inhibitors with Ki's ranging from 480 to 7900 microM. The inhibition of carboxypeptidase A by the alpha-monobrominated ketones is reversible and independent of the time (up to 6 h) of incubation of enzyme and inhibitor together. Bromoacetone at a concentration of 30 mM does not inhibit carboxypeptidase A. Incubation of an equimolar mixture of 2-benzyl-4-bromo-5-oxohexanoic acid (XV) and enzyme for 1 h led to the recovery of 82% of XV, demonstrating that it is the major species reversibly bound during assay of inhibition. Taken together, these results indicate that tight binding of carbonyl inhibitors to carboxypeptidase A requires specific binding of inhibitor functional groups such as benzyl and an electrophilic carbonyl carbon such as that of an alpha-bromo ketone or aliphatic aldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)     

Digestive proteinase activity of the Khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae)

The khapra beetle, Trogoderma granarium, is one of the most important stored product pests worldwide. A study of digestive proteinases in T. granarium was performed to identify potential targets for proteinaceous biopesticides, such as proteinase inhibitors. The pH of guts was determined by addition of pH indicator solutions to broken open gut regions. The last instar larvae were dissected in cold distilled water and the whole guts were cleaned from adhering unwanted tissues. The pooled gut homogenates were centrifuged and the supernatants were used in the subsequent enzyme assay. Total proteinases activity of the gut homogenates was determined using the protein substrate azocasein. Optimal azocasein hydrolysis by luminal proteinases of the larvae of T. granarium was highly alkaline in pH 10-10.5, although the pH of luminal contents was slightly acidic (pH 6.5). The extract showed the highest activity at 55 degrees C (pH 6.5), 45 degrees C (pH 8) and 30 degrees C (pH 10). The proteolytic activity was strongly inhibited in the presence of phenylmethylsulphonyl fluoride (82.33+/-4.37% inhibition). This inhibition was decreased with increasing of the pH of assay incubating medium. N-p-tosyl-L-lysine chloromethyl ketone (51.6+/-3.3% inhibition) and N-tosyl-L-phenylalanine chloromethyl ketone (27.23+/-4.37 % inhibition) showed inhibitory effect on proteolysis. Addition of thiol activators dithiothreitol and L-cysteine had not enhanced azocaseinolytic activity. The data suggest that protein digestion in the larvae of T. granarium is primarily dependent on serine proteinases; trypsin- and chymotrypsin-like proteinases.     

Partial purification and properties of D-desthiobiotin synthetase from Escherichia coli

d-Desthiobiotin synthetase, an enzyme that catalyzes the synthesis of d-desthiobiotin from dl-7,8-diaminopelargonic acid and HCO(3) (-), was purified 100-fold from cells of a biotin mutant strain of Escherichia coli. Adenosine triphosphate and Mg(2+) were shown, especially in purified extracts, to be obligatory for enzyme activity, although concentrations higher than 5 mm caused severe inhibition of the reaction with unpurified cell-free extracts. Adenosine diphosphate and adenosine monophosphate were shown to inhibit the reaction, but fluoride (up to 50 mm) had no detectable effect. The product of the enzyme reaction was identical to d-desthiobiotin on the basis of biological activity and chromatography. Furthermore, when H(14)CO(3) (-) was used as a substrate, the radioactive product was shown to be (14)C-desthiobiotin labeled exclusively in the ureido carbon.     

<Emphasis Type="Italic">Burkholderia cenocepacia</Emphasis>: a new biocatalyst for efficient bioreduction of ezetimibe intermediate

Ezetimibe is a selective acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor used in hypercholesterolemia. Synthesis of ezetimibe requires enantiopure 3-[5-(4-fluorophenyl)-5(S)-hydroxypentanoyl]-4(S)-4-phenyl-1,3-oxazolidin-2-one (FOP alcohol) as a crucial intermediate which is produced by reduction of the corresponding prochiral ketone (FOP dione). A new biocatalyst from acclimatized soil was screened for bioreduction of the above prochiral ketone. The microorganism was identified by 16S mRNA sequencing, as Burkholderia cenocepacia. Various physicochemical conditions were optimized to increase cellmass and enzyme activity. The overall increase in cellmass concentration and enzyme activity was 2.06 and 1.85-fold, respectively. Various reaction conditions, for example pH, temperature, agitation, and cellmass concentration, were optimized for maximum product yield (chiral alcohol) with excellent enantioselectivity. Best reduction was achieved in phosphate buffer (50 mM, pH 8.0) at 40°C (200 rpm) and the yield of enantiopure alcohol from the corresponding prochiral ketone was 54%. This biocatalyst was also used for the reduction of various other prochiral ketones.     

Adaptation of Clostridium tyrobutyricum for enhanced tolerance to butyric acid in a fibrous-bed bioreactor

By immobilization in a fibrous-bed bioreactor (FBB), we succeeded in adapting and selecting an acid-tolerant strain of Clostridium tyrobutyricum that can produce high concentrations of butyrate from glucose and xylose. This mutant grew well under high butyrate concentrations (>30 g/L) and had better fermentative ability as compared to the wild-type strain used to seed the bioreactor. Kinetic analysis of butyrate inhibition on cell growth, acid-forming enzymes, and ATPase activity showed that the adapted cells from the FBB are physiologically different from the original wild type. Compared to the wild type, the adapted culture's maximum specific growth rate increased by 2.3-fold and its growth tolerance to butyrate inhibition increased by 29-fold. The key enzymes in the butyrate-forming pathway, phosphotransbutyrylase (PTB) and butyrate kinase (BK), were also more active in the mutant, with 175% higher PTB and 146% higher BK activities. Also, the mutant's ATPase was less sensitive to inhibition by butyric acid, as indicated by a 4-fold increase in the inhibition rate constant, and was more resistant to the enzyme inhibitor N,N'-dicyclohexylcarbodiimide (DCCD). The lower ATPase sensitivity to butyrate inhibition might have contributed to the increased growth tolerance to butyrate inhibition, which also might be attributed to the higher percentage of saturated fatty acids in the membrane phospholipids (74% in the mutant vs 69% in the wild type). This study shows that cell immobilization in the FBB provides an effective means for in-process adaptation and selection of mutant with higher tolerance to inhibitory fermentation product.     

A note on the enzymic action and biosynthesis of a nitrile-hydratase from a Brevibacterium sp.

The effect of different compounds on the enzymic action of the nitrile-hydratase used for the bioconversion of nitriles was studied. An excess of acrylonitrile as a substrate was shown to inhibit the activity of the enzyme. This inhibition occurred only at relatively high substrate concentrations (0.2 mol/l or more). The nitrile bioconversion products (acrylamide, propionamide) and their structural analogues (acrylic acid, thioacetamide) were shown to inhibit the enzyme competitively. The most important inhibition found was that of cyanide (K_i= 0.004 mol/l), a break down product of some nitriles. By using an acetamidase-negative mutant, amides were shown to inhibit biosynthesis of nitrile-hydratase. An identical result was obtained with thioacetamide, a non-substrate compound for acetamidase. This compound repressed the biosynthesis of nitrile-hydratase by both the wild type and the acetamidase-negative mutant to the same extent.     

Biosynthesis of monoterpenes: demonstration of a geranyl pyrophosphate:(-)-bornyl pyrophosphate cyclase in soluble enzyme preparations from tansy (Tanacetum vulgare)

Tansy (Tanacetum vulgare L.) produces an essential oil containing the optically pure monoterpene ketone, (-)-camphor, as a major constituent. A soluble enzyme preparation from immature leaves of this plant converts the acyclic precursor [1-3H]geranyl pyrophosphate to the bicyclic monoterpene alcohol borneol in the presence of MgCl2, and oxidizes a portion of the borneol to camphor in the presence of a pyridine nucleotide. The identity of the major biosynthetic product as borneol was confirmed by chemical oxidation to camphor and crystallization of the derived oxime to constant specific radioactivity. The stereochemistry of the borneol was verified as the (-)-(1S,4S) isomer by oxidation to camphor, conversion to the corresponding ketal with D-(-)-2,3-butanediol, and separation of diastereoisomers by radio-gas-liquid chromatography. When enzyme reaction mixtures were treated with a mixture of acid phosphatase and apyrase, following an initial ether extraction of labeled borneol, additional quantities of borneol were generated, indicating the presence of a phosphorylated derivative of borneol. This water-soluble metabolite was prepared by large-scale enzyme incubations with [1-3H]geranyl pyrophosphate (plus phosphatase inhibitor), and the identity of the initial cyclization product was established as (-)-bornyl pyrophosphate by direct ion-exchange chromatographic analysis and enzymatic hydrolysis. The pathway for the formation of (-)-(1S,4S)-camphor was therefore identical to that previously demonstrated for the (+)-(1R,4R) isomer, involving cyclization of geranyl pyrophosphate to bornyl pyrophosphate, hydrolysis of this intermediate to borneol, and oxidation of the alcohol to the ketone. The labeling pattern of the product derived from [1-3H2, U-14C]geranyl pyrophosphate was determined by oxidation of the biosynthetic borneol to camphor and selective removal of tritium by exchange of the alpha hydrogens at C3 of the ketone. This labeling pattern was identical to that observed previously for the (+) isomer, suggesting the same mechanism of cyclization, but of opposite enantiospecificity. Some properties of the antipodal (+)- and (-)-bornyl pyrophosphate cyclases were compared.     

A new bacterial dehydrogenase oxidizing the lignin model compound guaiacylglycerol beta-O-4-guaiacyl ether

A lignin model compound, named in short guaiagylglycerol beta-guaiacyl ether (GGE), contains the beta-0-4 ether linkage that is common in the chemical structure of lignin. A Pseudomonas sp. (GU5) had been isolated as an organism able to grow with GGE as the sole source of carbon and energy. When grown on vanillate, the bacteria contained a NAD+ -dependent dehydrogenase converting GGE to a 355 nm absorbing product. The enzyme, named GGE-dehydrogenase, was purified about 160-fold using gel permeation, ion exchange on DEAE-Sephadex, and dye-ligand affinity chromatography. The new protein was about 52 kDa in apparent size with but one polypeptide chain after denaturation and reduction. According to several criteria, the product of GGE oxidation (Km = 12 microM) was identified as the corresponding conjugated ketone at the alpha-carbon of the C3 side-chain. The secondary alcohol function in GGE was apparently the sole target of the enzyme action. However the conversion of GGE into ketone catalyzed by the enzyme was only partial, and did not exceed 50%, probably because only one of the alpha-enantiomers was susceptible to enzyme attack. In contrast the ketone, either made by organic synthesis or by enzymic oxidation of GGE, could be totally reduced back to GGE (Km = 13 microM at pH 8.4, 8 microM at neutral pH), with NADH as the reductant, as confirmed by UV absorption and NMR spectra. Other model compounds with no primary alcoholic function, ether linkage or phenolic group were also substrates for the enzyme, confirming the specificity of GGE-dehydrogenase for the alpha-carbon position. Conjugation of the alpha-ketone with an adjacent phenolic nucleus interfered strongly with equilibrium constants and redox potentials of the system according to pH, and the enzyme displayed widely different optima with pH over 9 when oxidizing GGE, below 7 when reducing the ketone. Equilibrium studies showed that the ketone/GGE potential was -0.37 volt at pH 8.7, -0.23 volt at pH 7 (30 degrees C). The significance of this new dehydrogenase and its properties are discussed, especially in the general concern of lignin biodegradation.     

Kinetic mechanism of phosphofructokinase-2 from Escherichia coli. A mutant enzyme with a different mechanism

The kinetic mechanisms of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2 were investigated. Initial velocity studies showed that both enzymes have a sequential kinetic mechanism, indicating that both substrates must bind to the enzyme before any products are released. For Pfk-2, the product inhibition kinetics was as follows: fructose-1,6-P2 was a competitive inhibitor versus fructose-6-P at two ATP concentrations (0.1 and 0.4 mM), and noncompetitive versus ATP. The other product inhibition patterns, ADP versus either ATP or fructose-6-P were noncompetitive. Dead-end inhibition studies with an ATP analogue, adenylyl imidodiphosphate, showed uncompetitive inhibition when fructose-6-P was the varied substrate. For Pfk-2, the product inhibition studies revealed that ADP was a competitive inhibitor versus ATP at two fructose-6-P concentrations (0.05 and 0.5 mM), and noncompetitive versus fructose-6-P. The other product, fructose-1, 6-P2, showed noncompetitive inhibition versus both substrates, ATP and fructose-6-P. Sorbitol-6-P, a dead-end inhibitor, exhibited competitive inhibition versus fructose-6-P and uncompetitive versus ATP. These results are in accordance with an Ordered Bi Bi reaction mechanism for both enzymes. In the case of Pfk-2, fructose-6-P would be the first substrate to bind to the enzyme, and fructose-1,6-P2 the last product to be released. For Pfk-2, ATP would be the first substrate to bind to the enzyme, and APD the last product to be released.     

Selective Inhibition of Mutant Isocitrate Dehydrogenase 1 (IDH1) via Disruption of a Metal Binding Network by an Allosteric Small Molecule

Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg²⁺. A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors'' selectivity for the mutant enzyme.     

Kinetic studies on the two common inherited forms of human erythrocyte adenylate kinase

1. The kinetic properties of two genetic variants of human erythrocyte adenylate kinase were studied at limiting concentrations of both ADP and MgADP(-) in the forward direction and at limiting concentrations of both AMP and MgATP(2-) in the reverse direction. 2. Primary reciprocal plots rule out the possibility of a Ping Pong mechanism for both forms of the enzyme. 3. Analysis of the kinetic data by an appropriate computer program gave the following K(m) values for the type 1 enzyme: AMP, 0.33mm+/-0.1; MgATP(2-), 0.95mm+/-0.13; ADP, 0.12mm+/-0.03; MgADP(-), 0.22mm+/-0.04. Values for the type 2 enzyme were: AMP, 0.27mm+/-0.03; MgATP(2-), 0.40mm+/-0.05; ADP, 0.08mm+/-0.07; MgADP(-), 0.20mm+/-0.04. 4. Product inhibition studies were done by studying the reverse reaction. With ADP as product inhibitor competitive inhibition patterns were obtained with AMP and/or MgATP(2-) as variable substrate. Similar results were obtained for product inhibition by MgADP(-) with AMP as variable substrate. The results are consistent with a Rapid Equilibrium Random mechanism. 5. Secondary plots of slope versus product concentration were linear. The data were fitted to the appropriate equation and analysed by computer to give values for the product inhibition constants. 6. Differences between the values of certain kinetic constants for the two forms of the enzyme were observed.