Double-displacement Mechanism Research Articles

Inhibition of adenosine and thymidylate kinases by bisubstrate analogs.

Potential bisubstrate analogs, in which the 5'-hydroxyl group of adenosine was joined to the phosphoryl group acceptor by polyphosphoryl bridges of varying length (ApnX, where n is the number of phosphoryl groups and X is the nucleoside moiety of the acceptor), were tested as inhibitors of human liver adenosine kinase and of thymidylate kinase from peripheral blast cells of patients with acute myelocytic leukemia. Adenosine kinase was most strongly inhibited by P1,P4-(diadenosine 5')-tetraphosphate (Kd = 30 nM) and P1,P5-(diadenosine 5')-pentaphosphate (Kd = 73 nM). Thymidylate kinase was most strongly inhibited by P1-(adenosine 5')-P5-(thymidine 5')-pentaphosphate (Kd = 120 nM) and by P1(adenosine 5')-P6-(thymidine 5')-hexaphosphate (Kd = 43 nM). In these enzymes, as in adenylate and thymidylate kinases, strongest inhibition was achieved in compounds containing one or two more phosphoryl groups than the substrates combined. These results support the view that nucleoside and nucleotide kinases mediate direct transfer of phosphoryl groups from ATP to acceptors, rather than acting by a double displacement mechanism.

Catalysis by hog-kidney aminoacylase does not involve a covalent intermediate.

Hog kidney aminoacylase (N-acylamino acid amidohydrolase; acylase I) is shown to catalyze the exchange of acetate oxygens with water at a significant rate only when alanine is present simultaneously. These studies, conducted using the 18O-isotope induced shift on 13C-NMR spectra, provide evidence in favor of a linear kinetic mechanism as opposed to a 'ping-pong' double-displacement mechanism. At pH values above neutrality, aminoacylase I also catalyzes the exchange of alanine oxygens with those of water. Ionic strength and pH effects on the kinetics of aminoacylase I are examined and the results are interpreted in terms of a model of the enzyme active site.

Galactose-1-phosphate uridylyltransferase. Purification of the enzyme and stereochemical course of each step of the double-displacement mechanism.

A convenient new procedure for purifying galactose-1-phosphate uridylyltransferase from Escherichia coli is described. It departs from earlier methods by introducing the use of a Cibacron Blue-agarose (Bio-Rad Affi-Gel Blue) at an early stage. Purification is completed by ion-exchange chromatography using DEAE-Sephadex A-50. The procedure is substantially shorter than earlier methods and reproducibly yields enzyme of high specific activity suitable for use in structural work such as characterization of the intermediate uridylyl-enzyme. The first step of the galactose-1-P uridylyltransferase reaction is the transfer of the uridylyl group from UDP-glucose to N3 of a histidine residue in the enzyme to form the covalent uridylyl-enzyme and glucose-1-P. The uridylyl-enzyme intermediate then reacts in a second step with galactose-1-P to form UDP-galactose. The enzyme accepts (RP)-UDP alpha S-glucose as a good substrate, converting it to (RP)-UDP alpha S-galactose, i.e., with overall retention of configuration. In this paper we show that reaction of the enzyme with (RP)-[2-14C]UDP alpha S-glucose produces a [2-14C]uridylyl alpha S-enzyme that can be converted by base-catalyzed cyclization to (RP)-[2-14C]cUMPS. Inasmuch as cyclization must have proceeded with inversion of configuration at phosphorus, the corresponding configuration in the intermediate must have been the inverse of that in the substrate. Therefore, formation of uridylyl alpha S-enzyme from (RP)-UDP alpha S-glucose proceeds with inversion of configuration, and overall retention arises from inversion in each of the two steps. The results support the authenticity of the isolated uridylyl-enzyme as the true reaction intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterisation of sodium-dependent uptake of 5-hydroxytryptamine into guinea-pig brain

The uptake of 5-hydroxytryptamine (5-HT) was studied using isolated nerve endings (synaptosomes) prepared from the corpus striatum of the guinea pig. Analysis of temperature-dependent uptake revealed that two processes were involved, a sodium-dependent and sodium-independent mechanism. Kinetic studies indicated that the affinity of 5-HT ( K u) and the transport capacity ( V u) of the uptake process were dependent upon the external concentration of sodium. The transport capacity increased linearly with increasing concentrations of sodium up to 140 mmol/l, whereas the affinity for 5-HT was enhanced at concentrations of sodium up to 100 mmol/l, but reduced at 130 mmol/l sodium. The findings of this study suggest that uptake of 5-HT occurs as a result of a mobile carrier transport system which, depending upon the external concentration of sodium proceeds by a single or double displacement mechanism.

Cellular transglutaminase. The particulate-associated transglutaminase from chondrosarcoma and liver: partial purification and characterization.

A transglutaminase from the malignant chondrocytes, rat swarm chondrosarcoma cells, was partially purified and characterized in an effort to understand transformation-induced changes in its activity. This enzyme separated by DE52 column chromatography after extraction from the particulate fraction of cell lysate was found to be distinct from previously characterized transglutaminases in its electrophoretic mobility, molecular size, substrate specificity, and immunologic reactivity. This enzyme was identified as a transglutaminase by its catalysis of amine (putrescine, spermine) incorporation at the carboxamide group of protein-bound gamma-glutamyl residues, and accordance of its kinetic data with the modified double displacement mechanism described for other transglutaminases. Limited proteolysis of the isolated enzyme resulted in a 3-4-fold increase of catalytic activity and a concomitant reduction of molecular size by approximately one-half. Incubation of labeled amine with chondrosarcoma cell lysate resulted in labeling of only a few proteins that appeared to be extensively cross-linked and that were located mostly in the particulate fraction of the cells. Transglutaminase extracted from the rat liver particulate fraction displayed enzymatic and structural properties closely resembling those of the enzyme from chondrosarcoma cells.

A stereochemical study of the mechanism of activation of donor oligonucleotides by RNA ligase from bacteriophage T4 infected Escherichia coli.

RNA ligase from bacteriophage T4 infected Escherichia coli catalyzes the activation of adenosine 3',5'-bisphosphate (representing the donor oligonucleotide) by adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate with retention of configuration at P alpha. Since single-step enzyme-catalyzed nucleotidyl transfer reactions proceed with inversion, this stereochemical result provides support for a double displacement mechanism involving an adenylyl-enzyme intermediate as proposed previously from isotope exchange experiments.

Substrate specificity and stereospecificity of calf spleen phosphodiesterase towards deoxyribonucleosidyl 3'-(4-nitrophenyl phosphates) and phosphorothioates.

Phosphodiesterase from calf spleen exhibits nucleotidyltransferase activity when incubated with either the (PR) or the (PS) diastereomer of thymidyl 3'-(4-nitrophenyl phosphorothioate). Thymidylyl(3'-5')thymidyl phosphorothioate 3'-(4-nitrophenyl phosphorothioate) was identified as the main product of the enzyme-catalyzed reaction and the absolute configuration at the internucleotide phosphorus atom of the product was determined. The nucleotidyltransferase reaction is shown to proceed with retention of configuration at phosphorus, implying involvement of a double displacement mechanism with the formation of a nucleotidylated enzyme intermediate. To study the substrate specificity of spleen phosphodiesterase a series of deoxyribonucleosidyl 3'-(4-nitrophenyl phosphates) and phosphorothioates were synthesized, and Km and V parameters for each substrate were measured. The results obtained show virtually no specificity for substrates with different nucleosidyl moieties, while about a 20 - 30-fold drop in V and a slight increase in Km values is observed for phosphorothioate analogues as compared with corresponding phosphates. The enzyme showed no significant stereoselectivity towards phosphorothioates of opposite configurations at phosphorus.

Mechanistic implications of the inhibition of peptidases by amino aldehydes and bestatin

α-Amino aldehydes and bestatin are found to be effective inhibitors of a cytosolic dipeptidase (rat testicular peptidase C), and a cytosolic tripeptidase (rat kidney peptidase B, EC 3.4.11.4), as well as cytosolic leucine aminopeptidase (pig kidney peptidase S, EC 3.4.11.1). Aldehyde hydrates and bestatin share a resemblance to intermediates that might be formed during direct attack by water on peptide substrates, affording a possible explanation for their tight binding. Alternatively, inhibitors of both kinds might form derivatives of an active site nucleophile, resembling intermediates in a double-displacement mechanism. Exchange experiments with H 2 18O suggest that bestatin is bound intact by leucine aminopeptidase, lending support to the first of these two mechanisms.

Kinetic studies with the low-Km aldehyde reductase from ox brain.

Initial-rate studies of the low-Km aldehyde reductase-catalysed reduction of pyridine-3-aldehyde by NADPH gave families of parallel double-reciprocal plots, consistent with a double-displacement mechanism being obeyed. Studies on the variation of the initial velocity with the concentration of a mixture of the two substrates were also consistent with a double-displacement mechanism. In contrast, the initial-rate data indicated that a sequential mechanism was followed when NADH was used as the coenzyme. Product-inhibition studies, however, indicated that a compulsory-order mechanism was followed in which NADPH bound before pyridine-3-aldehyde with a ternary complex being formed and the release of pyrid-3-ylcarbinol before NADP+. The apparently parallel double-reciprocal plots obtained in the initial-rate studies with NADPH and pyridine-3-aldehyde were thus attributed to the apparent dissociation constant for the binary complex between the enzyme and coenzyme being finite but very low.

On the antiperiplanar lone pair hypothesis and its application to catalysis by glycosidases.

The idea that sp3 lone pairs of electrons on a heteroatom direct the departure of a leaving group from an adjacent tetrahedral carbon centre is shown to be untenable as a general principle, and the permissive evidence in favour of it to be accounted for by the principle of least nuclear motion. The application of the idea to the majority of glycopyranosidases, which work by a double-displacement mechanism, is shown to require implausible contortions of the pyranose ring.

Purification and some kinetic properties of rat liver ATP citrate lyase.

A new purification procedure for rat liver ATP citrate lyase is described. The method reproducibly gives homogenous undegraded enzyme. Steady-state kinetic analysis of ATP citrate lyase was complicated by the presence of ADP, a product of the reaction, in solutions of ATP. The kinetic patterns observed were dependent on whether ADP was removed by the assay system. When assays were performed with a method in which ADP was removed, the results showed that the enzyme obeys a double-displacement mechanism with a phosphoenzyme intermediate. This resolves a controversy between the results of previous kinetic studies and those of isotope-exchange and enzyme-labelling experiments.

De Novo Maltotriose Biosynthesis from the Reducing End by Spinacia oleracea L. Chloroplasts

The distribution of (14)C in the various glucose residues of maltotriose was studied as a function of time of photosynthesis of isolated chloroplasts of spinach (Spinacia oleracea L.) using (14)CO(2). The distribution of label showed that the reducing-end glucose residue was labeled first and the label subsequently distributed to the second and third glucose residues at approximately equal rates.A mechanism for the distribution of label and the synthesis of maltotriose from the reducing end is presented. The mechanism has postulated to be the same as that for the maltose synthase recently described by Schilling. Maltose biosynthesis from alpha-d-glucose-1-phosphate was characterized as involving two glucosyl intermediates by a double displacement mechanism with inversion of configuration. The mode of enzymic action by which maltosyl intermediates were transferred to glucosyl intermediates was consistent with the fractional distribution of radioactivity found in each glucose unit of maltotriose during short term photosynthesis experiments.

Rhodanese from Cercopithecus aethiops (vervet monkey) liver—II. Aspects of enzyme kinetics and mechanism of action

1. 1. Initial velocity kinetic studies were undertaken and certain kinetic parameters ( K m sso 2 3 − = 3.1 × 10 −3 M and V max = 153.85 U/ml/min.) were determined and the mechanism identified as a ping-pong (double displacement) mechanism. 2. 2. Competitive inhibition of rhodanese by both substrates, viz. thiosulphate and cyanide, provides additional evidence of Ping-Pong Bi-Bi mechanism for this transferase.

Kinetics and mechanism of erythrocyte anion exchange.

Steady state velocities for the rate of exchange of extracellular dithionite for intracellular sulfate through the red cell inorganic anion transport protein, known as band 3, were measured at increasing trans-sulfate concentrations at constant cis-sulfate concentration in order to directly discriminate between single and double displacement classes of mechanism. A 7-fold increase in trans-sulfate concentration resulted in intersecting Lineweaver-Burk plots under iso-osmotic conditions. This result argues against a double displacement or ping-pong mechanism. The specificity of the trans-anion effect for band 3 was illustrated by studying reversible 4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonate inhibition of anion exchange. The increase in trans-sulfate increased 4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonate affinity at the outer surface by about 4-fold under conditions where outside anion concentration was constant. A possible two-site ordered sequential mechanism for erythrocyte inorganic anion exchange is briefly discussed.

Stereochemical studies of enzyme-catalyzed alkyl-transfer reactions. An NMR method for distinguishing between the two prochiral hydrogens at C-1′ of spermidine

A simple NMR method is described that differentiates between the two prochiral hydrogen atoms at C-1′ of spermidine, thereby providing a means of distinguishing between a single- and double-displacement mechanism for the spermidine synthase reaction.

Alpha-aminoaldehydes: transition state analogue inhibitors of leucine aminopeptidase.

L-Leucinal, prepared by enzymatic oxidation of L-leucinol with alcohol dehydrogenase, is found to be a very strong competitive inhibitor of porcine kidney aminopeptidases. For the enzyme from kidney microsomes acting on L-leucine p-nitroanilide (Km = 5.2 x 10(-4) M), for Ki for L-leucinal was 7.6 x 10(-7) M at pH 7.2 and 25 degrees C. For the enzyme from kidney cytosol acting on L-leucine p-nitroanilide (Km = 7.7 x 10(-4) M), Ki for L-leucinal was 6 x 10(-8) M; Ki for glycinal (analogous to glycine derivatives that are poor substrates) was 6.8 x 10(-4) M. In dilute aqueous solution, leucinal exists in unfavorable equilibrium with its covalent hydrate, whose concentration exceeds that of the free aldehyde by a factor of 40. The affinity of the enzyme for the free aldehyde is correspondingly greater than its Ki values would suggest, exceeding the apparent affinity of the substrate by a factor of about 10(6). A comparison of binding affinities suggests that L-leucinal forms an inhibitory complex analogous in structure to unstable intermediates and substrate transformation by leucine aminopeptidase, and strengthens the likelihood that this enzyme may act by a double-displacement mechanism.

The stereochemical course of nucleotidyl transfer catalyzed by ATP sulfurylase.

ATP sulfurylase catalyzes the synthesis of ATP from adenosine 5'-phosphosulfate and magnesium pyrophosphate with inversion of configuration at phosphorus. This implies an "in line" displacement mechanism in the ternary complex and effectively eliminates both an adjacent mechanism followed by pseudorotation and a double displacement mechanism involving an adenylyl-enzyme intermediate. The double displacement mechanism had been invoked previously to account for a number of observations, including the ability of the enzyme to catalyze the hydrolysis of MgATP to AMP and MgPPi, and the exchange of Mg32PPi into MgATP in the absence of sulfate.

Inhibition of the puromycin reaction with benzamidine and related compounds

The similarity between the mode of action of proteolytic enzymes and ribosomal peptidyltransferasecatalyzed peptide bond formation is of considerable current interest [1,2]. Peptidyltransferase can function essentially in three possible ways: (i) Via a double displacement mechanism involving an acyl-enzyme (ribosome) intermediate as found in serine proteases [3 ]; (ii) By a direct transfer of peptidyl residue to aminoacyl function without forming a covalent ribosomal intermediate or catalytic participation of the ribosome [4]; or ('tii) Via a concerted cyclic mechanism which includes both transfer of peptidyl moiety and proton [5] from a suitably oriented functional group on the ribosome. One approach to an evaluation of possibility (i) is to study a response of some known inhibitors of proteolytic enzymes in a suitable model system of peptidyltransferase-catalyzed peptide bond formation. Thus, it was reported [6] that phenylboronic acids, a group of chymotrypsin irthibitors [7], inhibited the fragment reaction and also, to a lesser extent, the reaction of N-Ac-Phe-tRNA with puromycin. Unlike proteases, which are fairly specific for distinct amino acids, ribosomal peptidyltransferase is capable of catalyzing peptide-bond formation with aminoacyl tRNAs derived from all 20 amino acids. It is then likely that peptidyltransferase can incorporate the active site features of proteolytic enzymes other than simply those of chymotrypsin. Therefore, it was of interest to study a group of aromatic amidines, which are known to inhibit trypsin [8] as possible inhibitors of peptidyltransferase. All amidine derivatives, L-arginine and sodium benzoate, used here, were commercial products of the highest available purity./3-Phenylethylboronic acid was prepared as in [9,10]. The pH of all inhibitor solutions was adjusted to 7.4 with HC1 or NaOH prior to the assay. Inhibition of the puromycin reaction was performed as in [11 ]. A typical reaction mixture conrained in 0.1 ml: 0.05 M Tris-HC1 (pH 7.4), 0.1 M NH4CI, 0.01 M MgC12, 4.0 A26o units of NH4-washed 70 S ribosomes from E. coli MRE 600 cells, 10 g poly(U), 0.20 A26o units of N-Ac-[14C]Phe-tRNA (~5200 cpm, spec. act. 0.84 nmol [~4C]phenylalanine/mg tRNA), puromycin 1 X 10 -s M and inhibitor at desired concentrations. Following the incubation at 37°C for 30 mill, the reaction was stopped by addition of 0.1 ml 0.1 M Be(NOa)2 in 0.3 M acetate buffer (pH 5.5) saturated with MgSO4 and the products were extracted with 1.5 ml ethyl acetate. Ethyl acetate phase (1 ml) was transferred to a scintillation vial and the radioactivity was determined in 10 ml Scinti Verse TM scintillation mixture (Fisher Scientific Co., Fair Lawn N J) in a Packard Tri-Carb liquid scintillation spectrometer at 73% counting efficiency. Percent inhibition represents the difference in ethyl acetate extracted N-acetyl-L[ ~4 C] phenylalanylpuromycin counts in the absence and in the presence of inhibitors. Further experimental details are provided in the figure legends.

The stereochemical course of phosphoryl transfer catalysed by glucose 6-phosphatase.

Rat liver microsomal glucose 6-phosphatase catalyses phosphoryl transfer between D-glucose 6-[(R)-16O,17O,18O]phosphate and D-glucose with retention of configuration at the phosphorus atom. Since individual phosphoryl-transfer steps appear in general to occur with inversion of configuration, this observation is most simply interpreted in terms of a double-displacement mechanism with a phosphoryl-enzyme intermediate. Such an intermediate has been proposed previously from kinetic and 32P-labelling experiments.

2] Galactose-1-phosphate uridylyltransferase: Detection, isolation, and characterization of the uridylyl enzyme

This chapter discusses the isolation and characterization of the uridylyl enzyme, galactose-1-phosphate uridylyltransferase. Galactose-1-P uridylyltransferase can be purified from Escherichia coli, human erythrocytes, and Saccharomyces cerevisiae. Of these, the enzyme from E. coli is the most thoroughly characterized with respect to molecular properties and mechanism of action. While characterizing the catalytic pathway followed by E. coli galactose-1-P uridylyltransferase, the steady state rates are found to be consistent only with the double displacement (Ping Pong Bi Bi) kinetic model. The intermediate uridylyl-enzyme implied by the model given in the chapter can be isolated in both catalytically active and fully denatured forms. The uridylyl group in this intermediate has been shown to be bonded to N-3 of a histidine residue, and uridylyl group transfer has been shown to proceed with net retention of stereochemical configuration at Pα of substrates, which is consistent with a double-displacement mechanism.