Effects of noncovalent and covalent FAD binding on the redox and catalytic properties of p-cresol methylhydroxylase.

Each flavoprotein subunit (alpha or PchF) of the alpha(2)beta(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida contains FAD covalently attached to Tyr384. PCMH oxidizes p-cresol to 4-hydroxybenzyl alcohol, which is oxidized subsequently by PCMH to 4-hydroxybenzaldehyde. The Y384F mutant form of PchF (apo-PchF[Y384F]) displayed stoichiometric noncovalent FAD binding. PchF[Y384F]FAD associated with the cytochrome subunit (beta or PchC) (producing PCMH[Y384F]), although not as avidly as with wild-type PchF containing covalently bound FAD (PchF(C)). Dramatic increases in the two-electron E(m,7) (NHE) values for FAD were observed when it bound noncovalently to either apo-PchF or apo-PchF[Y384F], and the two-electron E(m,7) value for FAD was increased further by about 75 mV upon covalent binding to PchF, i.e., PchF(C). The E(m,7) values increased by approximately 20 and 45 mV, respectively, when PchF(C) and PchF[Y384F]FAD associated with PchC. The two-electron E(m,7) for covalently bound FAD in PCMH is 84 mV, the highest measured for a flavoprotein. The values for the one-electron redox potentials (E(m,7), NHE) for FAD were measured also for various forms of PchF. Under anaerobiosis, the reduction of PchF[Y384F]FAD by substrates was similar to that observed previously for PchF containing noncovalently bound FAD. Stopped-flow kinetic studies indicated a rapid substrate reduction of the FAD and heme in PCMH[Y384F] which produced PchF[Y384F]FAD(rad) x PchC, the mutant enzyme containing the flavin radical and reduced heme. These experiments also revealed a slow reduction of unassociated PchC(ox) by PchF[Y384F]FAD(rad) x PchC. Steady-state kinetic studies of the reaction of PCMH[Y384F] with p-cresol indicated that the K(m) for this substrate was unchanged relative to that of PCMH, but that the k(cat) was diminished by an order of magnitude. The data indicate that the covalent attachment of FAD to PchF assists catalysis by raising the E(m,7) of the flavin. Contributions to this effect likely result from conformational changes.

Heterologous expression in Pseudomonas aeruginosa and purification of the 9.2-kDa c-type cytochrome subunit of p-cresol methylhydroxylase.

The 9.2-kDa c-type cytochrome subunit (PchC) of the flavocytochrome p-cresol methylhydroxylase from Pseudomonas putida NCIMB 9869 has been overexpressed in recombinant form in Pseudomonas aeruginosa PAO1-LAC, using the recently developed pUCP-Nde vector. Efforts to produce the cytochrome in Escherichia coli using a pET vector, with or without its signal peptide, were generally unsuccessful, yielding relatively low levels of the protein. In contrast, the mature form of PchC accumulated in the periplasmic space of P. aeruginosa PAO1-LAC to about 1 mg/g wet cell paste. A periplasmic fraction enriched to about 12% (w/w) of total protein with recombinant PchC was isolated from the remainder of the cells by a washing procedure using ethylenediaminetetraacetate in the presence of sucrose. The cytochrome was purified to homogeneity from the periplasmic extract by anion-exchange chromatography on DEAE-Sepharose CL-6B followed by chromatofocusing on PolyBuffer Exchanger 94. Purified PchC was obtained in a yield of about 50% and was shown to be identical to that resolved from the native flavocytochrome isolated from P. putida. This system may prove to be of general use for the production of recombinant c-type cytochromes.

Structures of the flavocytochrome p-cresol methylhydroxylase and its enzyme-substrate complex: gated substrate entry and proton relays support the proposed catalytic mechanism.

The degradation of the toxic phenol p-cresol by Pseudomonas bacteria occurs by way of the protocatechuate metabolic pathway. The first enzyme in this pathway, p-cresol methylhydroxylase (PCMH), is a flavocytochrome c. The enzyme first catalyzes the oxidation of p-cresol to p-hydroxybenzyl alcohol, utilizing one atom of oxygen derived from water, and yielding one molecule of reduced FAD. The reducing electron equivalents are then passed one at a time from the flavin cofactor to the heme cofactor by intramolecular electron transfer, and subsequently to cytochrome oxidase within the periplasmic membrane via one or more soluble electron carrier proteins. The product, p-hydroxybenzyl alcohol, can also be oxidized by PCMH to yield p-hydroxybenzaldehyde. The fully refined X-ray crystal structure of PCMH in the native state has been obtained at 2. 5 A resolution on the basis of the gene sequence. The structure of the enzyme-substrate complex has also been refined, at 2.75 A resolution, and reveals significant conformational changes in the active site upon substrate binding. The active site for substrate oxidation is deeply buried in the interior of the PCMH molecule. A route for substrate access to the site has been identified and is shown to be governed by a swinging-gate mechanism. Two possible proton transfer pathways, that may assist in activating the substrate for nucleophilic attack and in removal of protons generated during the reaction, have been revealed. Hydrogen bonding interactions between the flavoprotein and cytochrome subunits that stabilize the intramolecular complex and may contribute to the electron transfer process have been identified.

Properties of p-cresol methylhydroxylase flavoprotein overproduced by Escherichia coli.

The alpha(2)beta(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida is composed of a flavoprotein homodimer (alpha(2) or PchF(2); M(r) = 119 kDa) with a cytochrome monomer (beta, PchC; M(r) = 9.3 kDa) bound to each PchF subunit. Escherichia coli BL21(DE3) has been transformed with a vector for expression of the pchF gene, and PchF is overproduced by this strain as the homodimer. During purification, it was recognized that some PchF had FAD bound, while the remainder was FAD-free. However, unlike PchF obtained from PCMH purified from P. putida, FAD was bound noncovalently. The FAD was conveniently removed from purified E. coli-expressed PchF by hydroxyapatite chromatography. Fluorescence quenching titration indicated that the affinity of apo-PchF for FAD was sufficiently high to prevent the determination of the dissociation constant. It was found that p-cresol was virtually incapable of reducing PchF with noncovalently bound FAD (PchF(NC)), whereas 4-hydroxybenzyl alcohol, the intermediate product of p-cresol oxidation by PCMH, reduced PchF(NC) fairly quickly. In contrast, p-cresol rapidly reduced PchF with covalently bound FAD (PchF(C)), but, unlike intact PCMH, which consumed 4 electron equiv/mol when titrated with p-cresol (2 electrons from p-cresol and 2 from 4-hydroxybenzyl alcohol), PchF(C) accepted only 2 electron equiv/mol. This is explained by extremely slow release of 4-hydroxybenzyl alcohol from reduced PchF(C). 4-Hydroxybenzyl alcohol rapidly reduced PchF(C), producing 4-hydroxybenzaldehyde. It was demonstrated that p-cresol has a charge-transfer interaction with FAD when bound to oxidized PchF(NC), whereas 4-bromophenol (a substrate analogue) and 4-hydroxybenzaldehyde have charge-transfer interactions with FAD when bound to either PchF(C) or PchF(NC). This is the first example of a "wild-type" flavoprotein, which normally has covalently bound flavin, to bind flavin noncovalently in a stable, redox-active manner.

Organization and sequences of p-hydroxybenzaldehyde dehydrogenase and other plasmid-encoded genes for early enzymes of the p-cresol degradative pathway in Pseudomonas putida NCIMB 9866 and 9869.

The gene (designated pchA) encoding the aldehyde dehydrogenase that is required to metabolise the p-hydroxybenzaldehyde produced by the degradation of p-cresol in Pseudomonas putida NCIMB 9866 and 9869 has been identified on plasmids pRA4000 and pRA500, respectively. The gene lies immediately upstream of the pchC and pchF genes encoding the subunits of p-cresol methylhydroxylase (PCMH), the preceeding enzyme in the p-cresol degradative pathway. In pRA500 the latter genes are followed by the genes encoding the alpha (pcaG) and beta (pcaH) subunits of protocatechuate-3,4-dioxygenase, whereas in pRA4000 the genes encoding PCMH are followed by an open reading frame encoding a protein that is similar to the maturase-related protein of P. alcaligenes. A gene, designated pchX, that encodes a protein of unknown function was identified between the pchC and pchF genes in both plasmids.

Expression of bovine activin-A and inhibin-A in recombinant baculovirus-infected Spodoptera frugiperda Sf 21 insect cells.

Currently, bioactive activin and inhibin for investigative purposes are obtained either by purification from bovine or porcine follicular fluid or have been kindly supplied in limited amounts by Genentech. The latter are recombinant formulations produced in cultured monkey kidney CV-1 cells. The aims of this study were to assess the potential of the baculovirus expression system as an alternative means to produce recombinant activin and inhibin. Towards these goals, two recombinant baculoviruses, AcBovACTA and AcBovINHA, were constructed. AcBovACTA contains a contiguous copy of the bovine beta A-inhibin/activin structural gene encoding the beta A-preproprotein whereas AcBovINHA contains contiguous copies of the bovine alpha-inhibin and beta A-inhibin/activin structural genes encoding the alpha- and beta A-preproproteins, respectively. Western blot analyses, using monoclonal antibodies specific for the mature portions of the alpha-inhibin and beta A-inhibin/activin subunits, demonstrated that Spodoptera frugiperda Sf21 cells infected with either recombinant virus secreted mature homodimeric activin-A into the medium. In addition, Sf21 cells infected with the recombinant AcBovINHA virus were found also to produce substantial amounts of the alpha-inhibin precursor protein. However, the mature portion of the latter is not secreted into the medium but is retained within infected cells in an incompletely processed form(s). The recombinant activin-A secreted by Sf21 cells infected with the AcBovACTA virus was shown to possess activin bioactivity when analysed by in vitor bioassay and, therefore, provides an alternative route to mammalian cell expression for the production of recombinant activin-A.

cDNA cloning of two splice variants of a human copper-containing monoamine oxidase pseudogene containing a dimeric Alu repeat sequence.

Two alternatively spliced transcripts, psiHLAO1 and psiHLAO2, of a copper-containing monoamine oxidase pseudogene have been isolated from a human-liver cDNA library. The larger psiHLAO1 cDNA (2073bp) contains a 5'-flanking segment of 134bp, followed by an apparent open reading frame (ORF) of 1725bp. The deduced amino acid sequence of this ORF (574 residues) shares 81.0% similarity with the 763-residue monoamine oxidase from human placenta (HPAO) (the N-terminal 533 residues of psiHLAO1 share 86.7% similarity with HPAO). The psiHLAO1 ORF is interrupted by an in-frame stop codon corresponding to amino acid 225 and terminates within a type S(a) dimeric Alu repeat sequence. psiHLAO2 appears to be an alternatively spliced variant of psiHLAO1 that has 413 bases of psiHLAO1 excised according to the 'GT-AG' rule. The slightly longer 3' end of the psiHLAO2 transcript shows that the Alu repeat is followed by an 11-bp poly(A) tract that, in turn, is followed by an AT-rich (81%) sequence of 105bp. A reverse transcriptase-polymerase chain reaction (RT-PCR) protocol was used to confirm that both psiHLAO1 and psiHLAO2 are transcribed in human liver and placenta. A search of the expressed sequence tag (EST) database indicates that, like HPAO, psiHLAO derives also from the region 17q21 of the human genome.

Redesign of choline acetyltransferase specificity by protein engineering.

Since the development of site-directed mutagenesis techniques over 15 years ago (Zoller, M. J., and Smith, M. (1982) Nucleic Acids Res. 10, 6487-6500), it has been a goal of protein engineering to utilize the procedure to redesign existing enzyme structures to produce proteins with altered or novel catalytic properties. To date, however, the more successful achievements have relied exclusively on the availability of three-dimensional protein structure maps to direct the redesign strategies. Presently, such maps are unavailable for choline acetyltransferase and carnitine acetyltransferase, enzymes that catalyze the reversible transfer of an acetyl group from acetyl-CoA to choline and L-carnitine, respectively. A more empirical approach, based on cross-referencing substrate structure comparisons with protein alignment data, was used to redesign choline acetyltransferase to accommodate L-carnitine as an acceptor of the acetyl group. A mutant choline acetyltransferase that incorporates four amino acid substitutions from wild type, shows a substantial increase in catalytic efficiency (kcat/Km) toward L-carnitine (1,620-fold) and shifts the catalytic discrimination between choline and L-carnitine by >390,000 in favor of the latter substrate. These dramatic alterations in catalytic function demonstrate that significant success in protein redesign can be achieved in the absence of three-dimensional protein structure data.

Isolation and characterization of an evolutionary precursor of human monoamine oxidases A and B.

An interesting flavoprotein-type monoamine oxidase (MAO) was recently isolated from Aspergillus niger and cloned [Schilling, B. & Lerch, K. (1995a) Biochim. Biophys. Acta 1243, 529-537; Schilling, B. & Lerch, K. (1995b) Mol. Gen. Genet. 247, 430-438]. The properties of this MAO, as well as a substantial part of its amino acid sequence, resemble those of both MAO A and B from higher animals, raising the possibility that it may be an evolutionary precursor of these mitochondrial enzymes. It differs from MAO A and B in several respects, however, including the fact that it is soluble and of peroxisomal location and that the FAD is non-covalently attached. We have overexpressed the fungal enzyme (MAO-N) in Escherichia coli and isolated it in pure form. Since several of the observations of previous workers on MAO-N could not be reproduced, we have reexamined its substrate specificity, interaction with reversible and irreversible inhibitors and other catalytic and molecular properties. MAO-N has a considerably higher turnover number on many aliphatic and aromatic amines than either form of the mammalian enzyme. Some aspects of the substrate specificity resemble those of MAO B, while others are similar to MAO A, including biphasic kinetics in double reciprocal plots. Contrary to a previous report [Schilling, B. & Lerch, K. (1995a) Biochim. Biophys. Acta 1243, 529-537], however, the fungal enzyme does not oxidize serotonin, norepinephrine, dopamine or other biogenic amines. MAO-N is irreversibly inhibited by stoichiometric amounts of both (-)deprenyl and clorgyline in a mechanism-based reaction, forming flavocyanine adducts with N5 of the FAD, like the mammalian enzymes, but inactivation is much faster with clorgyline than deprenyl, suggesting a closer resemblance to MAO A than B. The dissociation constants for a large number of reversible competitive inhibitors have been determined for MAO-N and comparison with similar values for MAO A and B again pointed to a greater similarity to the former than the latter.

The conserved serine-threonine-serine motif of the carnitine acyltransferases is involved in carnitine binding and transition-state stabilization: a site-directed mutagenesis study.

There has been speculation that the carnitine acyltransferase reaction mechanism may involve the formation of an acyl-serine intermediate. A serine-threonine-serine (STS) motif that is conserved throughout the carnitine acyltransferase family, and is present also in the choline acetyltransferases, contains the only two conserved serines. The functional role of this motif in carnitine octanoyltransferase was probed by using a site-directed mutagenesis strategy to generate all seven possible alanine substitutions: single, double and triple mutants. Kinetic analyses of these mutant enzymes demonstrated that the STS motif is not essential for catalysis, thereby excluding an acyl-serine intermediate from the reaction mechanism. The kinetic analyses support, however, substantial roles for the STS motif in carnitine binding and transition-state stabilization.

cDNA cloning, recombinant expression, and site-directed mutagenesis of bovine liver carnitine octanoyltransferase--Arg505 binds the carboxylate group of carnitine.

The cDNA for bovine liver carnitine octanoyltransferase (COT) has been cloned by a combination of lambda gt11 library screening and 3' rapid amplification of cDNA ends (3'-RACE). The cDNA comprises 338 bases of 5' non-coding sequence, a reading frame of 1839 bases including the stop codon, and 820 bases of 3' non-coding DNA. The deduced amino acid sequence of 612 residues predicts a protein with a calculated mass of 70263 Da and pI 6.28. The enzyme was expressed in recombinant soluble form in Escherichia coli and was purified by a two-step procedure to near-homogeneity with a yield of purified protein of 2-3 mg/l culture. Recombinant COT had similar kinetic properties to those of the enzyme isolated directly from beef liver. Arg505 in COT, conserved in all reported carnitine acyltransferase sequences but replaced by asparagine or isoleucine in the choline acetyltransferases, was converted to asparagine by site-directed mutagenesis. This single mutation resulted in a greater than 1650-fold increase in the Km value for COT towards carnitine, but had little effect on the value of k(cat) or the Km value for the acyl-CoA substrate. In addition, although choline was an extremely poor substrate for COT, the k(cat)/Km ratio towards this substrate was increased fourfold as a result of the mutation. These data support the notion that Arg505 in COT, and other carnitine acyltransferases, contributes to substrate binding by forming a salt bridge with the carboxylate moiety of carnitine.

Genomic cloning and sequence analyses of the bovine alpha-, beta A- and beta B-inhibin/activin genes. Identification of transcription factor AP-2-binding sites in the 5'-flanking regions by DNase I footprinting.

Inhibins and activins are dimeric peptide hormones that regulate the circulating levels of follicle-stimulating hormone (FSH). In turn, FSH stimulates inhibin gene expression in the ovarian follicle; studies to date suggest that this effect is mediated by cAMP and that a cAMP-responsive element, identified in the 5'-flanking region of the alpha-inhibin gene, at least partially effects this response. To explore further the transcriptional regulation of the inhibin/activin genes, we have isolated and sequenced the 5'-flanking regions of the bovine alpha-, beta A- and beta B-inhibin/activin subunit genes and have analysed these regions by primer-extension analysis and DNase I footprinting with the transcription factor AP-2. Analyses indicated that all three gene promoter regions have a number of AP-2-binding sites that are resistant to competition by poly(dI-dC), suggesting that cAMP may control the inhibin/activin ratio by operating through alternative signal-transduction pathways or that inhibin/activin gene expression may be controlled by signals operating through the protein kinase C pathway. A comparison of the DNA sequences protected by AP-2 against DNase I digestion revealed a consensus AP-2-binding site of 5'-GSCCCDSS-3', where S represents a base pairing involving three (C or G) hydrogen bonds and D represents any base other than C. The nucleotide sequences of the bovine beta-subunit structural genes also are reported.

The enzymes of the classical pentose phosphate pathway display differential activities in procyclic and bloodstream forms of Trypanosoma brucei.

The specific activities of each of the enzymes of the classical pentose phosphate pathway have been determined in both cultured procyclic and bloodstream forms of Trypanosoma brucei. Both forms contained glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconolactonase (EC 3.1.1.31), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ribose-5-phosphate isomerase (EC 5.3.1.6) and transaldolase (EC 2.2.1.2). However, ribulose-5-phosphate 3'-epimerase (EC 5.1.3.1) and transketolase (EC 2.2.1.1) activities were detectable only in procyclic forms. These results clearly demonstrate that both forms of T. brucei can metabolize glucose via the oxidative segment of the classical pentose phosphate pathway in order to produce D-ribose-5-phosphate for the synthesis of nucleic acids and reduced NADP for other synthetic reactions. However, only procyclic forms are capable of using the non-oxidative segment of the classical pentose phosphate pathway to cycle carbon between pentose and hexose phosphates in order to produce D-glyceraldehyde 3-phosphate as a net product of the pathway. Both forms lack the key gluconeogenic enzyme, fructose-bisphosphatase (EC 3.1.3.11). Consequently, neither form should be able to engage in gluconeogenesis nor should procyclic forms be able to return any of the glyceraldehyde 3-phosphate produced in the pentose phosphate pathway to glucose 6-phosphate. This last specific metabolic arrangement and the restriction of all but the terminal steps of glycolysis to the glycosome may be the observations required to explain the presence of distinct cytosolic and glycosomal isoenzymes of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. These same observations also may provide the basis for explaining the presence of cytosolic hexokinase and phosphoglucose isomerase without the presence of any cytosolic phosphofructokinase activity. The key enzymes of the Entner-Doudoroff pathway, 6-phosphogluconate dehydratase (EC 4.2.1.12) and 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) were not detected in either procyclic or bloodstream forms of T. brucei.

Role of arginine-292 in the substrate specificity of aspartate aminotransferase as examined by site-directed mutagenesis.

X-ray crystallographic data have implicated Arg-292 as the residue responsible for the preferred side-chain substrate specificity of aspartate aminotransferase. It forms a salt bridge with the beta or gamma carboxylate group of the substrate [Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H., & Christen, P. (1984) J. Mol. Biol. 174, 497-525]. In order to test this proposal and, in addition, to attempt to reverse the substrate charge specificity of this enzyme, Arg-292 has been converted to Asp-292 by site-directed mutagenesis. The activity (kcat/KM) of the mutant enzyme, R292D, toward the natural anionic substrates L-aspartate, L-glutamate, and alpha-ketoglutarate is depressed by over 5 orders of magnitude, whereas the activity toward the keto acid pyruvate and a number of aromatic and other neutral amino acids is reduced by only 2-9 fold. These results confirm the proposal that Arg-292 is critical for the rapid turnover of substrates bearing anionic side chains and show further that, apart from the desired alteration, no major perturbations of the remainder of the molecule have been made. The activity of R292D toward the cationic amino acids L-arginine, L-lysine, and L-ornithine is increased by 9-16-fold over that of wild type and the ratio (kcat/KM)cationic/(kcat/KM)anionic is in the range 2-40-fold for R292D, whereas this ratio has a range of [(0.3-6) x 10(-6)]-fold for wild type. Thus, the mutation has produced an inversion of the substrate charge specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

The roles of magnesium ions in the reaction catalysed by phosphofructokinase from Trypanosoma brucei.

The involvement of Mg2+ ions in the reaction catalysed by phosphofructokinase from Trypanosoma brucei was studied. The true substrate for the enzyme was shown to be the MgATP2-complex, and free Mg2+ ions are also required for enzyme activity. At concentrations of MgATP2- of 2.92 mM and greater, and a fructose 6-phosphate concentration of 1 mM and in the presence of EDTA as a Mg2+ buffer, the Km value for Mg2+ was determined to be 294 +/- 18 microM. Neither MgATP nor free ATP is an inhibitor of the enzyme, although apparent inhibition by the latter can be observed as a consequence of the decrease in free Mg2+ by chelation.

Kinetic studies on the reaction catalysed by phosphofructokinase from Trypanosoma brucei.

The steady-state kinetics of the reaction catalysed by the bloodstream form of Trypanosoma brucei were studied at pH 6.7. In the presence of 50 mM-potassium phosphate buffer, the apparent co-operativity with respect to fructose 6-phosphate and the non-linear relationship between initial velocity and enzyme concentration, which were found when the enzyme was assayed in 50 mM-imidazole buffer [Cronin & Tipton (1985) Biochem. J. 227, 113-124], are not evident. Studies on the variations of the initial rate with changing concentrations of MgATP and fructose 6-phosphate, the product inhibition by fructose 1,6-bisphosphate and the effects of the alternative substrate ITP were consistent with an ordered reaction pathway, in which MgATP binds to the enzyme before fructose 6-phosphate, and fructose 1,6-bisphosphate is the first product to dissociate from the ternary complex.

Purification and regulatory properties of phosphofructokinase from Trypanosoma (Trypanozoon) brucei brucei.

Phosphofructokinase (EC 2.7.1.11) from Trypanosoma (Trypanozoon) brucei brucei was purified to homogeneity by using a three-step procedure that may be performed within 1 day. Proteolysis, which removes a fragment of Mr approx. 2000, may occur during the purification, but this can be prevented by including antipain, an inhibitor of cysteine proteinases, in the buffers during the purification. The subunits of the enzyme appear to be identical in size, with an Mr of 49 000. The Mr of the native enzyme was estimated to be approx. 220 000, suggesting a tetrameric structure. Kinetic studies showed the activity to depend hyperbolically on the concentration of ATP but sigmoidally on the concentration of fructose 6-phosphate. Although cyclic AMP, AMP and ADP stimulated the enzyme activity at low concentrations of fructose 6-phosphate, the last two nucleotides were inhibitory at high concentrations of this substrate. Phosphoenolpyruvate behaved as an allosteric inhibitor of the phosphofructokinase. Citrate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and Pi did not influence significantly the activity of the enzyme.

Reaction of pig kidney aldehyde reductase with the coenzyme analogue 5'-p-fluorosulfonylbenzoyladenosine.

The kinetics of the interaction between the affinity label 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and the 'high Km' form of aldehyde reductase have been studied. The results of this investigation indicate the reaction to fulfill two of the major criteria of true affinity labelling (see Colman 1983), namely, that the reaction follows saturation kinetics, indicating the initial formation of a reversible enzyme-inhibitor complex, analagous to the enzyme-substrate complex, prior to the irreversible modification step, and that the interaction may be prevented specifically by the ligand (coenzyme in this case) which normally occupies the site towards which the label is directed. The amino acid(s) which binds covalently FSBA would thus be expected to be present within the coenzyme binding site of aldehyde reductase. The identification of the residue(s) attacked by this compound is currently under investigation.