Inhibition of microsomal oxidation by o-benzoquinone derivatives

It was found that o-benzoquinone derivatives inhibit the hydroxylation of aniline without any appreciable influence on the spectral changes which are due to cytochrome P-450 binding to the substrate. A comparative kinetic study of inhibition of aniline hydroxylation and NADPH-dependent reduction of o-benzoquinones in liver microsomes revealed that the inhibition is due to the ability of these compounds to shunt microsomal electron transport pathways.     

Oxidation of 4-anilino-5-methoxydioxybenzene-1,2 in complex biochemical systems possessing superoxide dismutase activity]

The kinetics of 4-anilino-5-methoxydioxybenzene-1,2 (AMOBQH2) autoxidation in biochemical systems possessing the superoxide dismutase activity were studied. The autoxidation of AMOBQH2 is affected by superoxide dismutase, which is indicative of participation of the superoxide radical in this process. The main kinetic effect of superoxide dismutase consists in a decrease of the effective rate constant for AMOBQH2 autoxidation. Peroxidase releases the superoxide dismutase inhibition of AMOBQH2 autoxidation. The data obtained are discussed in terms of a biochemical mechanism of action of biologically active aminoaromatic derivatives of o-benzoquinone.     

Effects of bile acids and their glycine conjugates on gamma-glutamyl transpeptidase

Glycine and taurine conjugates of bile acids modulate gamma-glutamyl transpeptidase by interacting with the cysteinylglycine binding site (acceptor site) of the enzyme. These compounds stimulate hydrolysis of glutamine and S-methylglutathione and the rate of the inactivation of the enzyme by the gamma-glutamyl site-directed reagent, AT-125 (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid). Transpeptidation between S-methylglutathione and methionine was inhibited by these compounds. These effects resemble those caused by hippurate; the glycine derivatives of bile acids, however, exhibit a much greater affinity for transpeptidase than hippurate. Cholate, as shown previously for benzoate, also seems to bind to a portion of the acceptor site as indicated by its effects on S-methylglutathione utilization and AT-125-dependent inactivation of the enzyme. The Kd values for cholate and benzoate are, however, at least one order of magnitude larger than those for their respective glycine derivatives. The acceptor site-directed modulators increase the affinity of the enzyme for AT-125 and kinetic and binding studies show that binding of gamma-glutamyl site-directed reagents increases the affinity of the enzyme for cholate. These results thus indicate cooperative interactions between the gamma-glutamyl donor and acceptor binding domains of the transpeptidase active center.     

The carbon paste electrode encrusted with a microreactor as glucose biosensor.

The amperometric biosensors based on carbon paste electrodes (CPEs) encrusted with single microreactor (MR) have been constructed for the determination of glucose. The MRs were prepared from CPC-silica carrier (CPC) and were loaded with glucose oxidase (GO), mediator (M) and acceptor (A). As the mediator cation radical of 5,10-dimethyl-5, 10-dihydrophenazine (DMDHP), N-methylphenazonium methyl sulfate (PMS) and o-benzoquinone (BQ) and as the acceptor Fe[EDTA]- or Fe(CN)6(3-) was used. The biosensors acted at electrode potential 0.15-0.27 V versus Ag-AgCl electrode. The calibration graphs of the biosensors were linear in the range from 1.5 to 50 mM of glucose. The sensitivity of the biosensors did not change at pH 6-8. The dissolved oxygen little (7%) influenced the biosensors response and 1 mM of ascorbic acid produced the response that was of equal value to 0.5 mM of glucose. The biosensors showed high stability; no change of the response of the biosensors prepared by using the novel microreactor was observed at least for 6 months by keeping the loaded CPC at room temperature in silica container. An optimization of the biosensors response against the GO, the mediator and the polymer amount was performed. The digital modeling of the biosensors action is following.     

Evaluation of benzofuroxan as a chromophoric oxidizing agent for thiol groups by using its reactions with papain, ficin, bromelain and low-molecular-weight thiols.

1. Benzofuroxan (benzofurazan 1-oxide, benzo-2-oxa-1,3-diazole N-oxide) was evaluated as a specific chromophoric oxidizing agent for thiol groups. 2. Aliphatic thiol groups both in low-molecular-weight molecules and in the enzymes papain (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4) readily reduce benzofuroxan to o-benzoquinone dixime; potential competing reactions of amino groups are negligibly slow. 3. The fate of the thiol depends on its structure: a mechanism is proposed in which the thiol and benzofuroxan form an adduct which, if steric factors permit, reacts with another molecule of thiol to form a disulphide; when the thiol is located in the active site of a thiol proteinase and steric factors preclude enzyme dinner formation, the adduct reacts instead with water or HO- to form a sulphenic acid; attack on the sulphur atom of the adduct by either a sulphur or oxygen nucleophile releases o-benzoquinone dioxine. 4. Benzofuroxan contains n o proton-binding sites with pKa values in the range 3-10 and probably none in the range 0-14; o-benzoquinone dioxine undergoes a one-proton ionization with pKa=6.75.5. o-benzoquinone dioxime absorbs strongly at wavelengths greater than 410nm, where absorption by benzofuroxan, proteins and simple thiol compounds is negligible; 416 nm is an isosbestic point (epsilon 416 = 5110 litre. mol-1-cm-1); epsilon430=3740+[1460/(1+[H+]/Ka)] where pKa=6.75. 6. The possibility of acid-base catalysis of the oxidation by active-centre histidine residues of the thiol proteinases is discussed.     

Kinetic mechanism of the aliphatic amidase from Pseudomonas aeruginosa.

The kinetic constants for hydrolysis and transfer (with hydroxylamine as the alternate acceptor) of the aliphatic amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were determined for a variety of acetyl and propionyl derivatives. The results obtained were consistent with a ping-pong or substitution mechanism. Product inhibition, which was pH dependent, implicated an acyl-enzyme compound as a compulsory intermediate and indicated that ammonia combined additionally with the free enzyme in a dead-end manner. The uncompetitive activation of acetamide hydrolysis by hydroxylamine and the observation that the partitioning of products between acetic acid and acetohydroxamate was linearly dependent on the hydroxylamine concentration substantiated these conclusions and indicated that deacylation was at least partially rate limiting. With propionamide as the acyl donor apparently anomalous results, which included inequalities in certain kinetic constants and a hyperbolic dependence of the partition ratio on the hydroxylamine concentration, could be explained by postulating a compulsory isomerisation of the acyl-enzyme intermediate prior to the transfer reaction.     

Benzofuroxan as a thiol-specific reactivity probe. Kinetics of its reactions with papain, ficin, bromelain and low-molecular-weight thiols.

1. The characteristics of benzofuroxan (benzofurazan 1-oxide, benzo-2-oxa-1,3-diazole N-oxide) that relate to its application as a reactivity probe for the study of environments of thiol groups are discussed. 2. To establish a kinetic and mechanistic basis for its use as a probe, a kinetic study of its reaction with 2-mercaptoethanol was carried out. 3. This reaction appears to proceed by a rate-determining attack of the thiolate ion on one of the electrophilic centres of benzofuroxan (possibly C-6) to provide a low steady-state concentration of an intermediate adduct; rapid reaction of this adduct with a second molecule of thiol gives the disulphide and o-benzoquinone dioxime. 4. The effects of the different types of environment that proteins can provide on the kinetic characteristics of reactions of thiol groups with benzofuroxan are delineated. 5. Benzofuroxan was used as a thiolspecific reactivity probe to investigate the active centres of papain (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). The results support the concept that the active centres of all three enzymes either contain a nucleophilic thiolate ion whose formation is characterized by a pKa of 3-4 and whose reaction with an electrophile can be assisted by interaction of a site of high electron density in the electrophile with active-centre imidazolium ion of pKa 8-9, or can provide such ions by protonic redistribution in enzyme-reagent or enzyme-substrate complexes.     

Initiation of chondroitin sulfate biosynthesis: a kinetic analysis of UDP-D-xylose: core protein beta-D-xylosyltransferase

The nature of the primary signals important for the addition of xylose to serines on the core protein of the cartilage chondroitin sulfate proteoglycan has been investigated. The importance of consensus sequence elements (Acidic-Acidic-Xxx-Ser-Gly-Xxx-Gly) in the natural acceptor was shown by the significant decrease in acceptor capability of peptide fragments derived by digestion of deglycosylated core protein with Staphylococcus aureus V8 protease, which cleaves within the consensus sequence, compared to the similar reactivity of trypsin-derived peptide fragments, in which consensus sequences remain intact. A comparison of the acceptor efficiencies (Vmax/Km) of synthetic peptides containing the proposed xylosylation consensus sequence and the natural acceptor (deglycosylated core protein) was then made by use of the in vitro xylosyltransferase assay. The two types of substrates were found to have nearly equivalent acceptor efficiencies and to be competitive inhibitors of each other's acceptor capability, with Km = Kiapparent. These results suggest that the artificial peptides containing the consensus sequence are analogues of individual substitution sites on the core protein and allowed the kinetic mechanism of the xylosyltransferase reaction to be investigated, with one of the artificial peptides as a model substrate. The most probable kinetic mechanism for the xylosyltransferase reaction was found to be an ordered single displacement with UDP-xylose as the leading substrate and the xylosylated peptide as the first product released. This represents the first reported formal kinetic mechanism for this glycosyltransferase and the only one reported for a nucleotide sugar:protein transferase.     

Estimation of intramolecular distance distributions in bovine pancreatic trypsin inhibitor by site-specific labeling and nonradiative excitation energy-transfer measurements

A series of four bovine pancreatic trypsin inhibitor (BPTI) derivatives, site specifically labeled by (2-methoxy-1-naphthyl)methyl (MNA) at the N-terminal amino group and by [7-(dimethylamino)-coumarin-4-yl]acetyl (DA-coum) at one of the four epsilon-amino groups, was prepared. The four derivatives, N alpha-MNA-Arg1-N epsilon-DA-coum-Lysn-BPTI [(1-n)BPTI] (n = 15, 26, 41, and 46), were purified by affinity chromatography and high-performance liquid chromatography (HPLC). The homogeneity of each derivative and its site of labeling were characterized by HPLC tryptic peptide mapping. Nonradiative energy transfer from MNA (donor) to DA-coum (acceptor) was measured by monitoring donor emission and acceptor excitation spectra. Transfer efficiencies between 45% and 85% were observed. The fluorescence decay of MNA in MNA-BPTI, a derivative labeled by a donor without an acceptor, is monoexponential, with a lifetime of 6.8 +/- 0.15 ns. The decay kinetics of MNA fluorescence measured for derivatives labeled both by donor and acceptor showed a small deviation from monoexponential decay with shorter average lifetimes. Analysis of the experimental decay curves yielded the detailed intramolecular distance distribution functions for each pair of labeled sites. The averages of the calculated distance distribution functions are close to the values expected from the known structure of BPTI in the crystalline state. The derivatives thus obtained are suitable for investigation of conformational transitions of the labeled protein and for monitoring localized changes such as those involved in the folding or unfolding transitions.     

From pyrroles to 1-oxo-2,3,4,9-tetrahydro-1H-beta-carbolines: a new class of orally bioavailable mGluR1 antagonists

Exploiting the SAR of the known pyrrole derivatives, a new class of mGluR1 antagonists was designed by replacement of the pyrrole core with an indole scaffold and consequent cyclization of the C-2 position into a tricyclic beta-carboline template. The appropriate exploration of the position C-6 with a combination of H-bond acceptor groups coupled with bulky/lipophilic moieties led to the discovery of a new series of mGluR1 antagonists. These compounds exhibited a non-competitive behavior, excellent pharmacokinetic properties, and good in vivo activity in animal models of acute and chronic pain, after oral administration.     

Dynamics and equilibrium for the formation of fluorescent Lewis acid-base exciplexes and triplexes between 9-aminophenanthrene and aliphatic amines.

The excited singlet states of 9-aminophenanthrene and its N-aminoalkyl derivatives are strongly fluorescent in cyclohexane. Addition of low concentrations of Et(3)N, Pr(2)NH, or PrNH(2) results in a red shift of the emission maximum and moderately decreased fluorescence intensity. Analysis of the fluorescence behavior using a combination of singular value decomposition with self-modeling and kinetic analysis provides evidence for the sequential formation of 1 : 1 (exciplex) and 1 : 2 (triplex) complexes between the excited 9-aminophenanthrene and ground-state alkylamine, both of which are strongly fluorescent. Both the formation and decay of the exciplex and triplex are dependent upon the extent of amine N-alkylation. Rate constants and equilibrium constants for complex formation follow the order 1 degree approximately 2 degree > 3 degree, analogous to that for the formation of ground-state complexes between amines and the soft Lewis base HgBr(2). Similarly, N-aminoalkyl derivatives of 9-aminophenanthrene form intramolecular exciplexes. Excited-state complex formation is attributed to a Lewis acid-base interaction between the excited aminophenanthrene (lone-pair acceptor) and ground-state amine (lone-pair donor). The factors which determine the stability of excited-state Lewis acid-base complexes are characteristic of the specific excited-state acceptor. No universal scale of lone-pair donor strength can be expected to describe the formation of such complexes.     

Kinetic basis for the donor nucleotide-sugar specificity of beta1, 4-N-acetylglucosaminyltransferase III

The kinetic basis of the donor substrate specificity of beta1, 4-N-acetylglucosaminyltransferase III (GnT-III) was investigated using a purified recombinant enzyme. The enzyme also transfers GalNAc and Glc moieties from their respective UDP-sugars to an acceptor at rates of 0.1-0.2% of that for GlcNAc, but Gal is not transferred at a detectable rate. Kinetic analyses revealed that these inefficient transfers, which are associated with the specificity of the enzyme, are due to the much lower V(max) values, whereas the K(m) values for UDP-GalNAc and UDP-Glc differ only slightly from that for UDP-GlcNAc. It was also found that various other nucleotide-Glc derivatives bind to the enzyme with comparable affinities to those of UDP-GlcNAc and UDP-Glc, although the derivatives do not serve as glycosyl donors. Thus, GnT-III does not appear to distinguish UDP-GlcNAc from other structurally similar nucleotide-sugars by specific binding in the ground state. These findings suggest that the specificity of GnT-III toward the nucleotide-sugar is determined during the catalytic process. This type of specificity may be efficient in preventing a possible mistransfer when other nucleotide-sugars are present in excess over the true donor.     

Purification and kinetic properties of lysophosphatidylinositol acyltransferase from bovine heart muscle microsomes and comparison with lysophosphatidylcholine acyltransferase

The enzyme acyl-CoA:1-acyl-sn-glycero-3-phosphoinositol acyltransferase (LPI acyltransferase, EC 2.3.1.23) was purified approximately 11,000-fold to near homogeneity from bovine heart muscle microsomes. The purification was effected by extraction with the detergent 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate, followed by chromatography on Cibacron blue agarose, DEAE-cellulose, and Matrex gel green A. The isolated enzyme was a single protein of 58,000 Da as measured by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. This purification procedure also allows isolation of the related enzyme lysophosphatidylcholine (LPC) acyltransferase, which was separated from LPI acyltransferase at the final chromatographic step. The purified LPI acyltransferase exhibits an absolute specificity for LPI as the acyl acceptor. Broader specificity was found for acyl-CoA derivatives as substrates, although the preferred substrates are long-chain, unsaturated derivatives: measured reactivities were in the order arachidonoyl-CoA greater than oleoyl-CoA greater than eicosadienoyl-CoA greater than linoleoyl-CoA. Little activity was found with palmitoyl-CoA or stearoyl-CoA as potential substrates. These properties are consistent with a role of the enzyme in controlling the acyl group composition of phosphoinositides. Comparison of LPC acyltransferase and LPI acyltransferase shows that these two enzymes have distinct kinetic and physical properties and are affected differently by local anesthetics, which are potent inhibitors.     

Kinetic studies of sheep kidney gamma-glutamyl transpeptidase.

The kinetics of sheep kidney gamma-glutamyl transpeptidase was studied using a novel substrate L-alpha-methyl-gamma-glutamyl-L-alpha-aminobutyrate. When the substrate was incubated with the enzyme in the presence of an amino acid or peptide acceptor, the corresponding L-alpha-methyl-gamma-glutamyl derivatives of the acceptors were formed. In the absence of acceptor only hydrolysis occurred, and no transpeptidation products were detected. The presence of the methyl group on the alpha-carbon apparently prevents enzymatic transfer of the L-alpha-methyl-gamma-glutamyl residue to the amino group of the substrate itself (autotranspeptidation). When the enzyme was incubated with conventional substrates, such as glutathione or gamma-glutamyl-p-nitroanilide and an amino acid acceptor, hydrolysis, autotranspeptidation, and transpeptidation to the acceptor occurred concurrently. Initial velocity measurements in which the concentration of L-alpha-methyl-gamma-glutamyl-L-alpha-aminobutyrate was varied at several fixed acceptor concentrations, and either the release of alpha-aminobutyrate or the formation of the transpeptidation products was determined, yielded results which are consistent with a ping-pong mechanism modified by a hydrolytic shunt. A scheme of such a mechanism is presented. This mechanism predicts the formation of an alpha-methyl-gamma-glutamyl-enzyme intermediate, which can react with an amino acid to form the transpeptidation product; or in the absence of, or in the presence of low concentrations of amino acids, can react with water to form the hydrolytic products. Kinetic derivations for the reaction of the enzyme with the conventional substrate gamma-glutamyl-p-nitroanilide predict either linear or nonlinear double-reciprocal plots, depending on the prevalence of the hydrolytic, autotranspeptidation, or transpeptidation reactions. The results of kinetic experiments confirmed these predictions.     

Some kinetic properties of gamma-glutamyltransferase from rabbit liver

gamma-Glutamyltransferase ((5-glutamyl)-peptide: amino-acid 5-glutamyltransferase, EC 2.3.2.2) of rabbit liver (detergent form) was purified 1100-fold in order to study its kinetic properties. Kinetic studies were conducted from pH 6.0 to 12.0 in the absence and presence of the acceptor substrate glycylglycine using gamma-glutamyl-3-carboxy-4-nitroanilide as the donor. The existence of more than one binding site for both donor and acceptor is postulated on kinetic evidence such as donor substrate activation, donor substrate inhibition and acceptor substrate activation. Homotropic interaction is also observed, in the form of negative cooperativity, in donor substrate binding, in the absence of acceptor at pH less than 9.0 and positive cooperativity (n = 2), in the absence or presence of acceptor at pH greater than 9.0. Hydrolase reaction reaches a maximum of activity at pH 10 (pK 8.6). Transferase activity under conditions of maximal velocity is maximal at pH 9.0 (pK 7.1). The ratio of transferase activity/hydrolase activity is maximal at pH 7.0-7.5. At low donor substrate concentrations, maximal activity is attained at pH 7.5.     

Active site studies of bovine alpha1-->3-galactosyltransferase and its secondary structure prediction

The catalytic domain of bovine alpha1-->3-galactosyltransferase (alpha3GalT), residues 80-368, have been cloned and expressed, in Escherichia coli. Using a sequential purification protocol involving a Ni(2+) affinity column followed by a UDP-hexanolamine affinity column, we have obtained a pure and active protein from the soluble fraction which catalyzes the transfer of galactose (Gal) from UDP-Gal to N-acetyllactosamine (LacNAc) with a specific activity of 0.69 pmol/min/ng. The secondary structural content of alpha3GalT protein was analyzed by Fourier transform infrared (FTIR) spectroscopy, which shows that the enzyme has about 35% beta-sheet and 22% alpha-helix. This predicted secondary structure content by FTIR spectroscopy was used in the protein sequence analysis algorithm, developed by the Biomolecular Engineering Research Center at Boston University and Tasc Inc., for the assignment of secondary structural elements to the amino acid sequence of alpha3GalT. The enzyme appears to have three major and three minor helices and five sheet-like structures. The studies on the acceptor substrate specificity of the enzyme, alpha3GalT, show that in addition to LacNAc, which is the natural substrate, the enzyme accepts various other disaccharides as substrates such as lactose and Gal derivatives, beta-O-methylgalactose and beta-D-thiogalactopyranoside, albeit with lower specific activities. There is an absolute requirement for Gal to be at the non-reducing end of the acceptor molecule which has to be beta1-->4-linked to a second residue that can be more diverse in structure. The kinetic parameters for four acceptor molecules were determined. Lactose binds and functions in a similar way as LacNAc. However, beta-O-methylgalactose and Gal do not bind as tightly as LacNAc or lactose, as their K(ia) and K(A) values indicate, suggesting that the second monosaccharide is critical for holding the acceptor molecule in place. The 2' and 4' hydroxyl groups of the receiving Gal moiety are important in binding. Even though there is large structural variability associated with the second residue of the acceptor molecule, there are constraints which do not allow certain Gal-R sugars to be good acceptors for the enzyme. The beta1-->4-linked residue at the second position of the acceptor molecule is preferred, but the interactions between the enzyme and the second residue are likely to be non-specific.     

Structural and kinetic analysis of substrate binding to the sialyltransferase Cst-II from Campylobacter jejuni

Sialic acids play important roles in various biological processes and typically terminate the oligosaccharide chains on the cell surfaces of a wide range of organisms, including mammals and bacteria. Their attachment is catalyzed by a set of sialyltransferases with defined specificities both for their acceptor sugars and the position of attachment. However, little is known of how this specificity is encoded. The structure of the bifunctional sialyltransferase Cst-II of the human pathogen Campylobacter jejuni in complex with CMP and the terminal trisaccharide of its natural acceptor (Neu5Ac-α-2,3-Gal-β-1,3-GalNAc) has been solved at 1.95 Å resolution, and its kinetic mechanism was shown to be iso-ordered Bi Bi, consistent with its dual acceptor substrate specificity. The trisaccharide acceptor is seen to bind to the active site of Cst-II through interactions primarily mediated by Asn-51, Tyr-81, and Arg-129. Kinetic and structural analyses of mutants modified at these positions indicate that these residues are critical for acceptor binding and catalysis, thereby providing significant new insight into the kinetic and catalytic mechanism, and acceptor specificity of this pathogen-encoded bifunctional GT-42 sialyltransferase.     

Phospholipase D-catalyzed synthesis of new phospholipids with polar head groups

A series of new phospholipids with polar head groups have been synthesized by enzymatic transphosphatidylation of 1,2-dioleoyl-sn-glycerophosphocholine and identified by 1H NMR and MALDI-TOF-MS. The acceptor alcohols were N- or C2-substituted derivatives of ethanolamine (diethanolamine, triethanolamine, serinol, Tris, BisTris). Phospholipases D from cabbage (PLDcab) and Streptomyces sp. (PLDStr) were compared with respect to product yield and purity as well as the initial rates in transphosphatidylation and competing hydrolysis. In all reactions, PLDStr showed a remarkably higher transphosphatidylation activity than PLDcab. However, higher yields of the phospholipids with diethanolamine, triethanolamine, and serinol were obtained by PLDcab because PLDStr resulted in the additional formation of diphosphatidyl derivatives. In the synthesis of the Tris and BisTris derivatives, PLD(Str) was much more appropriate because voluminous head group alcohols (>129A3) are poorly converted by PLDcab. With BisTris as acceptor alcohol two regioisomeric forms of phosphatidyl-BisTris were obtained.     

A series of site-specific fluorescently labeled BPTI derivatives prepared by nonselective acylation and chromatographic separations

Investigation of conformational transitions and, in particular, the folding/unfolding transitions of globular proteins by means of excitation energy transfer measurements depends on the availability of protein derivatives carrying donor and acceptor probes at well-defined pairs of sites. A series of bovine pancreatic trypsin inhibitor (BPTI) derivatives, each labeled at one of the epsilon-amino groups, was prepared. This was achieved by a nonselective acylation reaction using 7-dimethyl-amino-coumarin-4-acetyl-N-hydroxysuccinimide ester (DACA-NHSIE) as a reagent yielding a mixture of products. The mixture was resolved by affinity chromatography and reversed phase high performance liquid chromatography (HPLC). Four derivatives were obtained, each carrying the probe at one of the four amino groups. Identification of site of labeling and determination of the purity of the products was achieved by HPLC-tryptic peptide mapping. The labeled derivatives are active and can undergo a reversible denaturation/renaturation cycle. The spectral characteristics of the probe make it a suitable acceptor in energy-transfer measurements. The advantage of the approach described here, namely nonselective reaction combined with efficient fractionation procedures for the preparation of site specifically labeled derivatives, is that each of the amino groups can be labeled in a simple procedure, thus allowing for a maximal number of labeling sites which cannot be achieved when site-directed reagents (e.g. specific particular protection) are used. The present method yields derivatives which are useful in energy transfer measurements for determination of intramolecular distances between labeled sites. The derivatives should be useful in the analysis of the mechanism of protein folding and the intermediate structures involved.     

The development and application of a novel chromophoric substrate for investigation of the mechanism of yeast fatty acid synthase

The acetyl transacylase activity of the fatty acid synthase from yeast has been investigated using p-nitrophenylthiol acetate. The chromophoric nature of the nitrophenylthiol moiety affords a convenient spectrophotometric assay for the transacylase function as well as a means to investigate the kinetics and the mechanism of this process. A probable kinetic scheme for enzyme catalyzed transacetylation from p-nitrophenylthiol acetate to an acyl acceptor (CoA or N-acetylcysteamine) is proposed and the kinetic constants for acetylation of enzyme and for acetyl transfer to an acceptor were determined. It was also demonstrated that p-nitrophenylthiol acetate can replace acetyl-CoA as a substrate in fatty acid synthesis.