The inhibition of pepsin-catalysed reactions by products and product analogues. Kinetic evidence for ordered release of products

1. The inhibition of pepsin-catalysed hydrolysis of N-acetyl-l-phenylalanyl-l-phenylalanylglycine by products and product analogues was studied. 2. The non-competitive nature of the inhibition by the product N-acetyl-l-phenylalanine confirms an ordered release of products, and points to a common mechanism (involving an amino-enzyme) for pepsin-catalysed transpeptidation and hydrolysis reactions. 3. N-Acetyl-l-phenylalanine ethyl ester is also a non-competitive inhibitor, but here the inhibition is of the ;dead-end' type. No ethanol is detectable in reaction mixtures, indicating that this ester cannot act as an amino group acceptor in a transpeptidation process. 4. The same is true for N-methanesulphonyl-l-phenylalanine methyl and methyl thiol esters. No methanethiol is liberated when the methyl thiol ester is present as an inhibitor of the hydrolytic reaction, and the hope that such a thiol ester would effectively trap the amino-enzyme was not fulfilled.     

The pH-dependence of pepsin-catalysed reactions

1. The pH-dependence of the pepsin-catalysed hydrolysis of three peptide substrates was studied by using a method for the continuous monitoring of the formation of ninhydrin-positive products. 2. Two peptide acid substrates, N-acetyl-l-phenylalanyl-l-phenylalanine and N-acetyl-l-phenylalanyl-l-phenylalanyl-glycine, show apparent pK(a) values of 1.1 and 3.5 in the plots of k(0)/K(m) versus pH. By contrast a neutral substrate, N-acetyl-l-phenylalanyl-l-phenylalanine amide, shows apparent pK(a) values of 1.0 and 4.7. 3. Together with the data of the preceding paper (Knowles, Sharp & Greenwell, 1969), these results are taken to indicate that the rate of pepsin-catalysed hydrolysis is controlled by the ionization of two groups, which on the free enzyme have apparent pK(a) values of 1.0 and 4.7. It is apparent that the anions of peptide acid substrates are not perceptibly bound to the enzyme, resulting in apparent pK(a) values of 3.5 for the dependence of k(0)/K(m) for these materials.     

The rate-determining step in pepsin-catalysed reactions, and evidence against an acyl-enzyme intermediate

To delineate further the pathway of pepsin-catalysed reactions, three types of experiments were performed: (a) the enzyme-catalysed hydrolysis of a number of di- and tri-peptide substrates was studied with a view to observing the rate-determining breakdown of a common intermediate; (b) the interaction of pepsin with several possible substrates for which ;burst' kinetics might be expected was investigated; (c) attempts were made to trap a possible acyl-enzyme intermediate with [(14)C]methanol in both a hydrolytic reaction (with N-acetyl-l-phenylalanyl-l-phenylalanylglycine) and in a ;virtual' reaction (with N-acetyl-l-phenylalanine) under conditions where extensive hydrolysis or (18)O exchange is known to occur. It is concluded that (i) intermediates in pepsin-catalysed reactions (aside from the Michaelis complex) occur subsequently to the rate-determining transition state, and (ii) an acyl-enzyme intermediate, if such is formed, cannot be trapped with [(14)C]methanol in these systems.     

Acyl and amino intermediates in reactions catalysed by pig pepsin. Analysis of transpeptidation products.

The action of pig pepsin on a variety of small peptides including Leu-Trp-Met-Arg, Leu-Trp-Met, Leu-Leu-NH2, benzyloxycarbonyl-Phe-Leu and Gly-Leu-Tyr was studied. Leu-Leu-Leu was found to be the major product from the substrates Leu-Trp-Met-Arg and Leu-Trp-Met, indicating that the predominant reaction at pH 3.4 was a transpeptidation of the acyl-transfer type. Leu-Leu-Leu was also formed in high yield by amino transfer from benzyloxycarbonyl-Phe-Leu. Like the amino-transfer reactions the acyl transfer proceeded via a covalent intermediate, since [14C]leucine was not incorporated into transpeptidation products and did not exchange with enzyme-bound leucine in the presence of acceptors. With Leu-Trp-Met both acyl and amino transpeptidation products, namely Leu-Leu, Leu-Leu-Leu, Met-Met and Met-Met-Met, were formed in addition to methionine and leucine. With Leu-Trp-Met-Arg (1 mM) the pH optimum for the rates of hydrolysis and acyl transfer is about pH 3.4. At this pH the rate of acyl transfer exceeds that of hydrolysis; at pH 2, however, hydrolysis was faster than transfer. A comparison of the effect of the length of substrates and products on the reaction rates allows the conclusion that the binding site can extend over eight to nine amino acid residues. Although the experiments provide no conclusive evidence for or against the involvement of amino and/or acyl intermediates in the hydrolysis of long peptides and proteins, the high yield of transpeptidation reactions of both types observed with some substrates suggests a major role for the intermediates in pepsin-catalysed reactions. The results also show that when pig pepsin is used for the digestion of proteins for sequence work, the likelihood of the formation of transpeptidation products is considerable. In this way peptides not present in the original sequence could easily form in a reasonably good yield.     

Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis: I. Significance and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes

Achievement of efficient enzymatic degradation of cellulose to glucose is one of the main prerequisites and one of the main challenges in the biological conversion of lignocellulosic biomass to liquid fuels and other valuable products. The specific inhibitory interferences by cellobiose and glucose on enzyme-catalyzed cellulose hydrolysis reactions impose significant limitations on the efficiency of lignocellulose conversion — especially at high-biomass dry matter conditions. To provide the base for selecting the optimal reactor conditions, this paper reviews the reaction kinetics, mechanisms, and significance of this product inhibition, notably the cellobiose and glucose inhibition, on enzymatic cellulose hydrolysis. Particular emphasis is put on the distinct complexity of cellulose as a substrate, the multi-enzymatic nature of the cellulolytic degradation, and the particular features of cellulase inhibition mechanisms and kinetics. The data show that new strategies that place the bioreactor design at the center stage are required to alleviate the product inhibition and in turn to enhance the efficiency of enzymatic cellulose hydrolysis. Accomplishment of the enzymatic hydrolysis at medium substrate concentration in separate hydrolysis reactors that allow continuous glucose removal is proposed to be the way forward for obtaining feasible enzymatic degradation in lignocellulose processing.     

Phospholipase C-mediated hydrolysis of phosphatidylcholine is activated by muscarinic agonists.

The phospholipase C-catalysed breakdown of inositol-containing phospholipids is an important source of diacylglycerol in cells stimulated by several agonists. However, recent experimental evidence suggests that major phospholipids such as phosphatidylcholine may also be substrates of the phosphodiesteratic hydrolysis activated by hormones, growth factors and oncogene products. We show here that stimulation of muscarinic agonists activates the release of phosphocholine, which, along with diacylglycerol, is a metabolic product of phospholipase C-mediated hydrolysis of phosphatidylcholine. Fluoroaluminates mimic this muscarinic effect, strongly suggesting that carbachol-activated release of phosphocholine may be mediated by a guanine-nucleotide-binding protein. Evidence for this was obtained from experiments using permeabilized cells in which non-hydrolysable analogues of GTP activated phosphocholine release synergistically with carbachol.     

Kinetic and chemical mechanism of alpha-isopropylmalate synthase from Mycobacterium tuberculosis

Mycobacterium tuberculosis alpha-isopropylmalate synthase (MtIPMS) catalyzes the condensation of acetyl-coenzyme A (AcCoA) with alpha-ketoisovalerate (alpha-KIV) and the subsequent hydrolysis of alpha-isopropylmalyl-CoA to generate the products CoA and alpha-isopropylmalate (alpha-IPM). This is the first committed step in l-leucine biosynthesis. We have purified recombinant MtIPMS and characterized it using a combination of steady-state kinetics, isotope effects, isotopic labeling, and (1)H-NMR spectroscopy. The alpha-keto acid specificity of the enzyme is narrow, and the acyl-CoA specificity is absolute for AcCoA. In the absence of alpha-KIV, MtIPMS does not enolize the alpha protons of AcCoA but slowly hydrolyzes acyl-CoA analogues. Initial velocity studies, product inhibition, and dead-end inhibition studies indicate that MtIPMS follows a nonrapid equilibrium random bi-bi kinetic mechanism, with a preferred pathway to the ternary complex. MtIPMS requires two catalytic bases for maximal activity (both with pK(a) values of ca. 6.7), and we suggest that one catalyzes deprotonation and enolization of AcCoA and the other activates the water molecule involved in the hydrolysis of alpha-isopropylmalyl-CoA. Primary deuterium and solvent kinetic isotope effects indicate that there is a step after chemistry that is rate-limiting, although, with poor substrates such as pyruvate, hydrolysis becomes partially rate-limiting. Our data is inconsistent with the suggestion that a metal-bound water is involved in hydrolysis. Finally, our results indicate that the hydrolysis of alpha-isopropylmalyl-CoA is direct, without the formation of a cyclic anhydride intermediate. On the basis of these results, a chemical mechanism for the MtIPMS-catalyzed reaction is proposed.     

Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis: II. Quantification of inhibition and suitability of membrane reactors

Product inhibition of cellulolytic enzymes affects the efficiency of the biocatalytic conversion of lignocellulosic biomass to ethanol and other valuable products. New strategies that focus on reactor designs encompassing product removal, notably glucose removal, during enzymatic cellulose conversion are required for alleviation of glucose product inhibition. Supported by numerous calculations this review assesses the quantitative aspects of glucose product inhibition on enzyme-catalyzed cellulose degradation rates. The significance of glucose product inhibition on dimensioning of different ideal reactor types, i.e. batch, continuous stirred, and plug-flow, is illustrated quantitatively by modeling different extents of cellulose conversion at different reaction conditions. The main operational challenges of membrane reactors for lignocellulose conversion are highlighted. Key membrane reactor features, including system set-up, dilution rate, glucose output profile, and the problem of cellobiose are examined to illustrate the quantitative significance of the glucose product inhibition and the total glucose concentration on the cellulolytic conversion rate. Comprehensive overviews of the available literature data for glucose removal by membranes and for cellulose enzyme stability in membrane reactors are given. The treatise clearly shows that membrane reactors allowing continuous, complete, glucose removal during enzymatic cellulose hydrolysis, can provide for both higher cellulose hydrolysis rates and higher enzyme usage efficiency (kg_product/kg_enzyme). Current membrane reactor designs are however not feasible for large scale operations. The report emphasizes that the industrial realization of cellulosic ethanol requires more focus on the operational feasibility within the different hydrolysis reactor designs, notably for membrane reactors, to achieve efficient enzyme-catalyzed cellulose degradation.     

Kinetic studies of enzymatic hydrolysis of insoluble cellulose: (II). Analysis of extended hydrolysis times

The kinetics of enzymatic hydrolysis of pure insoluble cellulose by means of unpurified culture filtrate of Trichoderma reesei was studied, emphasizing the kinetic characteristics associated with the extended hydrolysis times. The changes in the hydrolysis rate and extent of soluble protein adsorption during the progress of reaction, either apparent or intrinsic, were investigated. The hydrolysis rate declined drastically during the initial hours of hydrolysis. The factors causing the reduction in the hydrolysis rate were examined; these include the transformation of cellulose into a less digestible form and product inhibition. The structural transformation can be partially explained by changes in the crystallinity index and surface area. The product inhibition was caused by the deactivation of the adsorbed soluble protein by the products, which essentially represents the so-called "un-competitive" inhibition. The kinetics of beta-glucosidase were also studied. The result has shown that the action of beta-glucosidase is competitively inhibited by glucose. It has been found that the integrated form of the initial rate expression cannot be used in predicting the progress of reaction because the digestibility of cellulose changes drastically as the hydrolysis proceeds, and that the rate expression for enzymatic hydrolysis of cellulose cannot be simplified or approximated by resorting to the pseudo-steady-state assumption. A mechanistic kinetic model of cellulose hydrolysis should include the following major influencing factors: (1)mode of action of enzyme, (2) structure of cellulose, and (3) mode of interaction between the enzyme and cellulose molecules.     

Proton-Nuclear Magnetic Resonance Analyses of the Substrate Specificity of a β-Ketolase from Pseudomonas putida, Acetopyruvate Hydrolase

A revised purification of acetopyruvate hydrolase from orcinol-grown Pseudomonas putida ORC is described. This carbon-carbon bond hydrolase, which is the last inducible enzyme of the orcinol catabolic pathway, is monomeric with a molecular size of approximately 38 kDa; it hydrolyzes acetopyruvate to equimolar quantities of acetate and pyruvate. We have previously described the aqueous-solution structures of acetopyruvate at pH 7.5 and several synthesized analogues by (1)H-nuclear magnetic resonance (NMR)-Fourier transform (FT) experiments. Three (1)H signals (2.2 to 2.4 ppm) of the methyl group are assigned unambiguously to the carboxylate anions of 2,4-diketo, 2-enol-4-keto, and 2-hydrate-4-keto forms (40:50:10). A (1)H-NMR assay for acetopyruvate hydrolase was used to study the kinetics and stoichiometries of reactions within a single reaction mixture (0.7 ml) by monitoring the three methyl-group signals of acetopyruvate and of the products acetate and pyruvate. Examination of 4-tert-butyl-2,4-diketobutanoate hydrolysis by the same method allowed the conclusion that it is the carboxylate 2-enol form(s) or carbanion(s) that is the actual substrate(s) of hydrolysis. Substrate analogues of 2,4-diketobutanoate with 4-phenyl or 4-benzyl groups are very poor substrates for the enzyme, whereas the 4-cyclohexyl analogue is readily hydrolyzed. In aqueous solution, the arene analogues do not form a stable 2-enol structure but exist principally as a delocalized pi-electron system in conjugation with the aromatic ring. The effects of several divalent metal ions on solution structures were studied, and a tentative conclusion that the enol forms are coordinated to Mg(2+) bound to the enzyme was made. (1)H-(2)H exchange reactions showed the complete, fast equilibration of (2)H into the C-3 of acetopyruvate chemically; this accounts for the appearance of (2)H in the product pyruvate. The C-3 of the product pyruvate was similarly labelled, but this exchange was only enzyme catalyzed; the methyl group of acetate did not undergo an exchange reaction. The unexpected preference for bulky 4-alkyl-group analogues is discussed in an evolutionary context for carbon-carbon bond hydrolases. Routine one-dimensional (1)H-NMR in normal (1)H(2)O is a new method for rapid, noninvasive assays of enzymic activities to obtain the kinetics and stoichiometries of reactions in single reaction mixtures. Assessments of the solution structures of both substrates and products are also shown.     

Aliphatic Amidase from <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> M8 Is Related to the Nitrilase/Cyanide Hydratase Family

A comparative study of amino acid sequence and physicochemical properties indicates the affiliation of an amidase from Rhodococcus rhodochrous M8 (EC 3.5.1.4) to the nitrilase/cyanide hydratase family. Cluster analysis and multiple alignments show that Cys166 is an active site nucleophile. The enzyme has been shown to be a typical aliphatic amidase, being the most active toward short-chain linear amides. Small polar molecules such as hydroxylamine and O-methyl hydroxylamine can serve as effective external nucleophiles in acyl transfer reactions. The kinetics of the industrially important amidase-catalyzed acrylamide hydrolysis has been studied over a wide range of substrate concentrations; inhibition during enzymatic hydrolysis by the substrate and product (acrylic acid) has been observed; an adequate kinetic scheme has been evaluated and the corresponding kinetic parameters have been determined.     

Integral kinetics of alpha-galactosidase purified from Glycine max for simultaneous hydrolysis of stachyose and raffinose

alpha-Galactosidase from soybean (Glycine max) was purified by a five-step procedure. The enzyme's natural substrates, raffinose and stachyose, have K(m)'s of 3. 0 mM and 4. 79 mM, respectively. The products, galactose and sucrose, were measured after separation by liquid chromatography. Galactose is a competitive product inhibitor of stachyose and raffinose hydrolysis with a K(i) of 0. 12 mM. We determined these parameters by an integral kinetic approach. Stachyose hydrolysis gives a nearly constant level of raffinose shortly after hydrolysis begins. Thus, cleavage of the first alpha-(1,6)-bond in the tetrasaccharide is the rate-limiting step. Since the stachyose hydrolysis yields raffinose, soybean alpha-galactosidase simultaneously hydrolyzes two substrates. We present a novel approach for analyzing simultaneous substrate hydrolysis with competitive product inhibition by a modified integral rate expression. The experimentally found kinetic parameters are confirmed by solving the simultaneous equations which describe the hydrolysis. This technique may be applicable to other hydrolytic enzymes with multiple substrates.     

Synthesis and Nuclear Magnetic Resonance Spectroscopic, Mass Spectroscopic, and Plasma Amine Oxidase Inhibitory Properties of Analogues of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine

Abstract: Seventeen analogues of l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine were synthesized using three reaction pathways: condensation of phenols with 1-methyl-4-piperidone, reaction of Grignard reagents with 1-methyl-4-piperidone followed by dehydration of the product, and aminomethylation of olefins. The identity of the products of synthesis was established by nuclear magnetic resonance spectroscopy, mass spectroscopy. and elemental analysis. Thirteen analogues were shown to inhibit the oxidation of benzylamine by bovine plasma amine oxidase. Increasing the length of the aliphatic chain of N -substituted analogues resulted in increased inhibition. In 4-phenyl-substituted analogues, both the position and electronic character of the substituent group affected the degree of inhibition.     

Glycoprotein synthesis in maize endosperm cells: the nucleoside diphosphate-sugar: dolichol-phosphate glycosyltransferases

Microsomal membrane preparations from maize (Zea mays L., inbred A636) endosperm cultures contained enzymes that transferred sugar moieties from uridine diphosphate-N-acetylglucosamine, guanosine diphosphate-mannose, and uridine diphosphate-glucose to dolichol-phosphate. These enzyme activities were characterized with respect to detergent and pH optima, substrate kinetic constants, and product and antibiotic inhibition constants. It was demonstrated by mild acid hydrolysis and high performance liquid chromatography that the products of the N-acetylglucosamine transferases were N-acetylglucosamine-pyrophosphoryl-dolichol and N,N′-diacetyl-chitobiosyl-pyrophosphoryl-dolichol and that the product of the mannose transferase was mannosyl-phosphoryl-dolichol. A large proportion of the products of the glucose transferase activity was stable to mild acid hydrolysis. However, the proportion that was labile was identified as glucosyl-phosphoryl-dolichol. Rate zonal sedimentation and isopycnic banding in linear sucrose density gradients in the presence of 1 millimolar ethylenediaminetetraacetic acid indicated that the glycosyltransferase activities were located in the endoplasmic reticulum. The glycosyltransferases were not solubilized by 500 millimolar KCl or by sequential washes with tris-(hydroxymethyl)aminomethane and water, treatments that release peripheral membrane proteins. Solubilization was achieved with low concentrations of Triton X-100. When sealed microsomal vesicles were incubated with trypsin for 30 minutes in absence of detergent, the activity of N-acetylglucosaminyl-transferase was substantially reduced, while the activity of the glucosyl-transferase was somewhat reduced. Activity of the mannosyl-transferase was resistant to inactivation by incubation with trypsin unless Triton was present.     

Structure-activity relationships in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta

The mechanism and substrate specificity of the phosphotriesterase from Pseudomonas diminuta have been examined. The enzyme hydrolyzes a large number of phosphotriester substrates in addition to paraoxon (diethyl p-nitrophenyl phosphate) and its thiophosphate analogue, parathion. The two ethyl groups in paraoxon can be changed to propyl and butyl groups, but the maximal velocity and Km values decrease substantially. The enzyme will not hydrolyze phosphomonoesters or -diesters. There is a linear correlation between enzymatic activity and the pKa of the phenolic leaving group for 16 paraoxon analogues. The beta value in the corresponding Br?nsted plot is -0.8. No effect on either Vmax or Vmax/Km is observed when sucrose is used to increase the relative solvent viscosity by 3-fold. These results are consistent with rate-limiting phosphorus-oxygen bond cleavage. A plot of log V versus pH for the hydrolysis of paraoxon shows one enzymatic group that must be unprotonated for activity with a pKa of 6.1. The deuterium isotope effect by D2O on Vmax and Vmax/Km is 2.4 and 1.2, respectively, and the proton inventory is linear, which indicates that only one proton is "in flight" during the transition state. The inhibition patterns by the products are consistent with a random kinetic mechanism.     

Pyranonaphthoquinone derivatives of eleutherin,ventiloquinone L,thysanone and nanaomycin A possessing a diverse topoisomerase II inhibition and cytotoxicity spectrum

A series of pyranonaphthoquinone derivatives related to the known topoisomerase II inhibitor eleutherin 1 have been shown to act as specific topoisomerase II catalytic inhibitors, with several analogues displaying greater potency than the natural product itself. Amongst the compounds tested were the natural products ventiloquinone L 4 and thysanone 8 with a diverse range of topoisomerase II inhibition properties being observed. Interestingly, the natural products are generally weaker inhibitors than their synthetic counterparts, emphasising that subtle changes in the basic molecular structure of a natural product led to significant changes in the inhibition profile. It has also been demonstrated for the first time that analogues related to nanaomycin A and cardinalin-type dimeric pyranonaphthoquinones exhibit potent topoisomerase II inhibitory properties. With respect to structural features, it appears that the nature of the substituents at C1 on the pyran ring and oxygenated substituents on the aryl ring are critical for anti-topoII activity.Importantly, the topoisomerase II inhibition strength does not correlate well with the measured cytotoxicity against yeast, indicating that other molecular features in the pyranonaphthoquinone family must be considered for the design and use of this structural class as highly specific topoisomerase II inhibitors.     

Kinetic study of the action of snake venom phospholipase A2 on human serum high density lipoprotein 3.

The hydrolysis of the phospholipids of intact human serum high density lipoprotein 3 (HDL3) by pure alpha-phospholipase A2 from Crotalus adamanteus was studied by pH-stat titration. The enzyme quantitatively hydrolyzed phosphatidylcholine and phosphatidylethanolamine and left sphinogomyelin intact, yielding a stable and water-soluble modified HDL. Lysophospholipids and free fatty acids, the products of hydrolysis, remained in the lipoprotein. When 1 mol of defatted bovine serum albumin/mol of substrate phospholipids was added to the reaction mixture, up to 60% of the fatty acids and 85% of the lysophospholipids were removed from the modified lipoprotein. The immunological reactivity of the hydrolyzed HDL remained unaltered in both the presence and absence of albumin. The changes in the physical properties of the lipoprotein during hydrolysis were rather small, the most notable being an increase in the hydrated density and in the electrophoretic mobility in alkaline buffers. The hydrolysis followed an apparent first order time course with product inhibition (KI) and yielded values of kcat/Km = 7 X 10(5 M(-1)s(-1) and KI congruent to 1 X 10(-4) M. Addition of albumin to the reaction mixture relieved the product inhibition without any alteration of the kinetic parameters. High concentrations of albumin protected some of the substrate phospholipids from hydrolysis, presumably through complexation to the lipoprotein. The Arrhenius plot for the experimental first order rate constant in the absence of albumin (kexp = kcat (KI/Km)) was linear between 15 degrees and 47 degrees, indicating the absence of any phospholipid phase transitions and yielding an activation energy of 15.2 kcal/mol. From the accessibility of the HDL phospholipids to phospholipase A2 one concludes that the phosphatidylcholine and phosphatidylethanolamine are located at, or are in rapid equilibrium with, the surface of this lipoprotein. It also appears that these phospholipids are not essential for maintaining the supramolecular properties of the lipoprotein in vitro. Thsu the study of the modified Hdl should provide valuable information concenring the structure and function of this lipoprotein particularly with regard to the role played by shiingomyelin.     

Bioconversion of 2-hydroxy-6-oxo-6-(4'-chlorophenyl)hexa-2,4-dienoic acid, the meta-cleavage product of 4-chlorobiphenyl

Bacterial conversion of 4-chlorobiphenyl (4-CB) usually proceeds through a pathway involving an initial oxidation of the unsubstituted ring in the 2,3 position followed by a 1,2 meta-cleavage. The meta-cleavage product (MCP) is converted through a single hydrolysis step into chlorobenzoic acid. However, several other acidic metabolites that were not expected as part of this pathway have already been described. In this paper, we used strains of Pseudomonas putida carrying cloned genes from Pseudomonas testosteroni B-356 that are involved in polychlorinated biphenyl (PCB) degradation to demonstrate that several acidic metabolites found in the culture media of various bacteria grown in the presence of 4-CB result from alternative novel bioconversion pathways of MCP. The degradation products of MCP through these pathways were identified as analogues with saturated or shorter side chains or as 4'-chlorophenyl-2-picolinic acid; pathways leading to their formation are proposed.     

Mechanism of the prenyl-transfer reaction. Studies with (E)- and (Z)-3-trifluoromethyl-2-buten-1-yl pyrophosphate

The prenyl-transfer reaction catalyzed by porcine farnesyl pyrophosphate synthetase has been studied using (E)- and (Z)-3-trifluoromethyl-2-buten-1-yl pyrophosphates as substrates and inhibitors. The rate of condensation between isopentenyl pyrophosphate (IPP) and the allylic fluoro analogues is drastically depressed relative to the normal catalytic rate observed with dimethylallyl pyrophosphate (DMAPP) or geranyl pyrophosphate (GPP). A similar depression is found in the rates of solvolysis for methanesulfonate derivatives of the fluoro analogues in aqueous actone under typical SN1 reaction conditions. Prolonged incubation of [14C] IPP and (E)- or (Z)-CF3-DMAPP with the enzyme, followed by treatment with alkaline phosphatase, gave a product that comigrated with geranylgeraniol on a polystyrene column. Both fluoro analogues showed mixed linear inhibition patterns with DMAPP or GPP as the variable substrate. We interpret these results in terms of an ionization-condensation-elimination mechanism for the prenyl-transfer reaction.