Kinetic investigation of the substrate specificity of the cyanogenic beta-D-glucosidase (linamarase) of white clover

Partially purified linamarase from Trifolium repens (genotype Lili acac) plants was kinetically characterized. Kinetic evidence was found to support the assumption that this cyanogenic beta-D-glucosidase has a broad substrate spectrum. p-Nitrophenyl-beta-D-xylopyranoside and p-nitrophenyl-alpha-L-arabinopyranoside substrates bound almost as tightly to the active center of the enzyme as the glucono(1----5)lactone transition-state analog inhibitor. Substrate specificity investigation also indicated that positions C-4 and C-6 on the pyranoside ring play an essential role in both substrate orientation and splitting. Recently very similar kinetic characteristics were reported on some mammalian cytosolic beta-D-glucosidases and a possible physiological interpretation of this coincidence is discussed. Inhibition studies with glucono(1----5)lactone revealed that the carbohydrate moiety of each substrate attached to the same binding site in the active center. Inhibition experiments with 1-thio substrate analogs demonstrated that the aglycon and the angular arrangement around the glycosidic linkage were the major determinants in the observed substrate specificity.     

Physical and kinetic properties of a plasma-membrane-bound beta-D-glucosidase (cellobiase) from midgut cells of an insect (Rhynchosciara americana larva).

The midgut caecal cells from Rhynchosciara americana larvae possess a plasma-membrane-bound beta-D-glucosidase (cellobiase, EC 3.2.1.21), which is recovered (75-95%) in soluble form both after treatment with Triton X-100 and after treatment with papain. The Triton X-100-solubilized beta-D-glucosidase displays Mr106000 and pI 5.4, whereas the papain-released beta-D-glucosidase shows Mr65000 and pI 4.7. Thermal inactivations of the detergent-solubilized and the papain-released forms of beta-D-glucosidase both follow apparent first-order kinetics with similar half-lives. The papain-released beta-D-glucosidase, after being purified by density-gradient centrifugation, hydrolyses beta-D-glucosides, beta-D-galactosides and beta-D-fucosides at the same active site, as inferred from experiments of competition between substrates. The beta-D-glucosidase seems to operate in accordance with rapid-equilibrium kinetics, since the Km (0.61 mM) for the enzyme is constant over a wide range of pH. The hydrolysis of the beta-D-glucosidic bond catalysed by the beta-D-glucosidase occurs without inversion of configuration, delta-gluconolactone is a strong (Ki 0.5 microM) inhibitor of the enzyme and substituents in the substrate aglycone affect the catalytic constant of the reaction. These data support the assumption that the mechanism of the reaction catalysed by the beta-D-glucosidase involves the intermediary formation of a carbonium ion, rather than a glucosyl-enzyme intermediate.     

Structural determinants of substrate specificity in family 1 beta-glucosidases: novel insights from the crystal structure of sorghum dhurrinase-1, a plant beta-glucosidase with strict specificity, in complex with its natural substrate

Plant beta-glucosidases play a crucial role in defense against pests. They cleave, with variable specificity, beta-glucosides to release toxic aglycone moieties. The Sorghum bicolor beta-glucosidase isoenzyme Dhr1 has a strict specificity for its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-beta-D-glucoside), whereas its close homolog, the maize beta-glucosidase isoenzyme Glu1, which shares 72% sequence identity, hydrolyzes a broad spectrum of substrates in addition to its natural substrate 2-O-beta-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxaxin-3-one. Structural data from enzyme.substrate complexes of Dhr1 show that the mode of aglycone binding differs from that previously observed in the homologous maize enzyme. Specifically, the data suggest that Asn(259), Phe(261), and Ser(462), located in the aglycone-binding site of S. bicolor Dhr1, are crucial for aglycone recognition and binding. The tight binding of the aglycone moiety of dhurrin promotes the stabilization of the reaction intermediate in which the glycone moiety is in a deformed (1)S(3) conformation within the glycone-binding site, ready for nucleophilic attack to occur. Compared with the broad specificity maize beta-glucosidase, this different binding mode explains the narrow specificity of sorghum dhurrinase-1.     

Mapping of the active site of rat kidney gamma-glutamyl transpeptidase using activated esters and their amide derivatives

The enzyme gamma-glutamyl transpeptidase (GGT), implicated in many physiological processes, catalyses the transfer of a gamma-glutamyl from a donor substrate to an acyl acceptor substrate, usually an amino acid or a peptide. In order to investigate which moieties of the donor substrate are necessary for recognition by GGT, the structure of the well-recognized substrate L-gamma-glutamyl-p-nitroanilide was modified. Several activated esters and their amide derivatives were synthesized and used as substrates. Kinetic (K(m) and V(max)) and inhibition constants (K(i)) were measured and reveal that almost the entire gamma-glutamyl moiety is necessary for recognition in the binding site of the donor substrate. The implied presence of certain complementary amino acids in this substrate binding site will allow the more rational design of various substrate analogues and inhibitors.     

Binary and ternary complexes of malate dehydrogenase with substrates and substrate analogs

Hydroxypyrenetrisulfonate binds to pig mitochondrial malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) in the presence and absence of coenzymes with a stoichiometry of one dye molecule/enzyme subunit. Binding is competitive with substrates and known substrate analogs as well as with squaric acid, a newly detected analog forming a ternary complex with enzyme/NAD+ similar to enzyme/NAD+/sulfite. Displacement of hydroxypyrenetrisulfonate by substrates and analogs was used to determine dissociation constants of binary and ternary complexes. Binary complexes form with dissociation constants of about 10 mM. They may be important for kinetic studies at high substrate concentrations where oxaloacetate inhibition and malate activation have been described.     

Human liver sulphamate sulphohydrolase. Determinations of native protein and subunit Mr values and influence of substrate agylcone structure on catalytic properties.

Human sulphamate sulphohydrolase was purified at least 20,000-fold to homogeneity from liver with a three-step four-column procedure, which consisted of a concanavalin A-Sepharose/Blue A agarose coupled step, and Bio-Gel HT step and then a CM-Sepharose step. The procedure was also used to purify enzyme from kidney and placenta. The subunit Mr of liver, kidney and placenta sulphamate sulphohydrolase was assessed to be 56,000 by using SDS/polacrylamide-gel electrophoresis. The native protein Mr of enzyme from all three tissue sources was assessed by gel-permeation chromatography to be approx. 120,000 on Sephacryl S-300 and 100,000 on Fractogel TSK. It is probable that the native enzyme results from dimerization of subunits. Kinetic parameters (km and kcat.) of human liver sulphamate sulphohydrolase were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparin and heparan sulphate. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, are turned over up to 372000 times faster than the monosaccharide substrate 2-sulphaminoglucosamine. Aglycone structures that influence substrate binding and/or enzyme activity were penultimate-residue C-6 carboxy and C-2 sulphate ester groups and a post-penultimate 2-sulphaminoglucosamine residue. The C-4 hydroxy group of the 2-sulphaminoglucosamine under enzymic attack is involved in binding of substrate to enzyme. The presence of C-6 sulphate ester on the non-reducing end 2-sulphaminoglucosamine stimulates sulphamate bond hydrolysis and substrate affinity if the adjacent monosaccharide residue is idose or 2-sulphoidose, but strongly inhibits hydrolysis if the adjacent monosaccharide residue is iduronic acid. Sulphamate sulphohydrolase is an exoenzyme, since activity toward internal sulphamate bonds was not detected. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure. The presence of aglycone C-2 sulphate ester and aglycone C-6 carboxy groups and C-6 sulphate ester groups on the 2-sulphaminoglucosamine residue under attack considerably affect the pH response. Structurally complex substrates had two pH optima. Incubation temperature and buffer ionic strength markedly influenced pH optima and enzyme activity. Cu2+ and SO4(2-)ions are potent inhibitors of enzyme activity.     

Nucleotide regulation of Escherichia coli glycerol kinase: initial-velocity and substrate binding studies

Substrate binding to Escherichia coli glycerol kinase (EC 2.7.1.30; ATP-glycerol 3-phosphotransferase) was investigated by using both kinetics and binding methods. Initial-velocity studies in both reaction directions show a sequential kinetic mechanism with apparent substrate activation by ATP and substrate inhibition by ADP. In addition, the Michaelis constants differ greatly from the substrate dissociation constants. Results of product inhibition studies and dead-end inhibition studies using 5'-adenylyl imidodiphosphate show the enzyme has a random kinetic mechanism, which is consistent with the observed formation of binary complexes with all the substrates and the glycerol-independent MgATPase activity of the enzyme. Dissociation constants for substrate binding determined by using ligand protection from inactivation by N-ethylmaleimide agree with those estimated from the initial-velocity studies. Determinations of substrate binding stoichiometry by equilibrium dialysis show half-of-the-sites binding for ATP, ADP, and glycerol. Thus, the regulation by nucleotides does not appear to reflect binding at a separate regulatory site. The random kinetic mechanism obviates the need to postulate such a site to explain the formation of binary complexes with the nucleotides. The observed stoichiometry is consistent with a model for the nucleotide regulatory behavior in which the dimer is the enzyme form present in the assay and its subunits display different substrate binding affinities. Several properties of the enzyme are consistent with negative cooperativity as the basis for the difference in affinities. The possible physiological importance of the regulatory behavior with respect to ATP is considered.     

Inhibition of H(+)-transporting ATPase, Ca(2+)-transporting ATPase and H+/K(+)-transporting ATPase by strophanthidin.

Studies of the effect of strophanthidin on H(+)-transporting ATPase, Ca(2+)-transporting ATPase and H+/K(+)-transporting ATPase activities are reported. Inhibition observations and kinetic results suggest the existence of a common digitalis aglycone binding site located on the extracellular surface of the enzyme, which is affected competitively by the binding of potassium to H(+)-transporting ATPase, Ca(2+)-transporting ATPase, as well as H+/K(+)-transporting ATPase and Na+/K(+)-transporting ATPase. This may lead to a better understanding of the mechanism of the pharmacological action of cardiac glycosides and imply the possibility that the positive inotropic effect may result from the inhibition of both Ca(2+)-transporting ATPase and Na+/K(+)-transporting ATPase.     

Substrate and inhibitor studies of thermolysin-like neutral metalloendopeptidase from kidney membrane fractions. Comparison with bacterial thermolysin

The inhibitory constants of a series of synthetic N-carboxymethyl peptide inhibitors and the kinetic parameters (Km, kcat, and kcat/Km) of a series of model synthetic substrates were determined for the membrane-bound kidney metalloendopeptidase isolated from rabbit kidney and compared with those of bacterial thermolysin. The two enzymes show striking similarities with respect to structural requirements for substrate binding to the hydrophobic pocket at the S1' subsite of the active site. Both enzymes showed the highest reaction rates with substrates having leucine residues in this position while phenylalanine residues gave the lowest Km. The two enzymes were also inhibited by the same N-carboxymethyl peptide inhibitors. Although the mammalian enzyme was more susceptible to inhibition than its bacterial counterpart, structural variations in the inhibitor molecules affected the inhibitory constants for both enzymes in a similar manner. The two enzymes differed significantly, however, with respect to the effect of structural changes in the P1 and P2' positions of the substrate on the kinetic parameters of the reaction. The mammalian enzyme showed the highest reaction rates and specificity constants with substrates having the sequence -Phe-Gly-Phe- or -Phe-Ala-Phe- in positions P2, P1, and P1', respectively, while the sequence -Ala-Phe-Phe- was the most favored by the bacterial enzyme. The sequence -Gly-Gly-Phe- as found in enkephalins was not favored by either of the enzymes. Of the substrates having an aminobenzoate group in the P2' position, the mammalian enzyme favored those with the carboxyl group in the meta position while the bacterial enzyme favored those with the carboxyl group in the para position.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel pronounced reversible inhibition of phosphofructokinase, glucose 6-phosphate dehydrogenase, and phosphoglucose isomerase by hexacyanoferrate (II).

At pH 6.8, pig kidney phosphofructokinase (PFK) is inhibited 90% by 1 mm hexacyanoferrate(II), in a reaction mixture containing 0.2 mm fructose 6-phosphate (F-6-P) and 1 mm ATP. Glucose 6-phosphate dehydrogenase and phosphoglucose isomerase are inhibited 70% by 5 mm hexacyanoferrate(II), at a 0.2 mm concentration of their respective substrates. Unlike all previously reported inhibitions of glycolytic enzymes by hexacyanoferrate, this inhibition seems not to involve an oxidation of enzyme, substrate, or enzyme-substrate complex. It appears to be due to reversible binding of the hexacyanoferrate at, or near, the hexose phosphate binding site of each enzyme. These inhibition studies were carried out in 50 mm 2-mercaptoethanol, and spectral studies showed that these conditions ensured that all the hexacyanoferrate was in the reduced (II) state. The inhibition of PFK was competitive with respect to the substrate F-6-P. Some reaction between hexacyanoferrate(II) and the substrate could not be definitely ruled out, but such reactions cannot be the major basis for the inhibitions observed. Increasing the magnesium concentration did not overcome the PFK inhibition. For all three enzymes, addition of a high concentration of hexose phosphate substrate to an assay mixture containing highly inhibited enzyme resulted in removal of the inhibition. The inhibition was instantaneous, and there was no increase in inhibition with time of incubation with hexacyanoferrate(II). These results may provide an approach to active-site labeling of these three enzymes at their hexose phosphate binding sites. These results should also be of interest to other workers, especially those involved in oxidative phosphorylation studies, who use ferro- and ferricyanide as research tools. The effects from such experiments may, in some cases, be due to binding of these compounds at, or near, hexose phosphate binding sites in the system.     

Acetylcholinesterase. Two types of inhibition by an organophosphorus compound: one the formation of phosphorylated enzyme and the other analogous to inhibition by substrate

1. The kinetics of the reaction of di-(2-chloroethyl) 3-chloro-4-methylcoumarin-7-yl phosphate (haloxon) and related compounds with acetylcholinesterase were studied and found to be unusual. 2. By a progressive reaction haloxon produces a di-(2-chloroethyl)phosphorylated enzyme. The influence of substrate on this reaction leading to a phosphorylated active centre was studied. From competition experiments between inhibitor and substrate values of K(m) for acetylcholine and acetylthiocholine of 0.79mm and 0.23mm respectively were derived. 3. Haloxon also combines with acetylcholinesterase by a non-progressive reaction, producing a complex that is reversible by dilution and by high concentrations of acetylcholine and acetylthiocholine. From this non-progressive reaction the competition between haloxon and substrate was studied, and it was shown that haloxon combines with a site involved in inhibition by substrate. From competition experiments the following dissociation constants were derived: for combination of haloxon and this site K(i) is 4.9mum and for the combination of substrates with this site K(88) values are 12mm and 3.3mm for acetylcholine and acetylthiocholine respectively. 4. The non-phosphorus-containing compound 3-chloro-7-hydroxy-4-methylcoumarin was shown to be a good reagent for the site involved in inhibition by substrate; its dissociation constant for the combination with this site is 30mum. 5. In order to interpret the experimental results, theoretical equations were derived for an enzyme with two binding sites to both of which substrate and inhibitor can combine. The equations correlate the activity of the enzyme with the concentration of substrate and inhibitor, for both progressive and non-progressive inhibition. These equations are applicable to reactions of acetylcholinesterase with organophosphorus compounds, carbamates etc. and may be applicable to other enzymes possessing two binding sites.     

The crystal structure of human cytosolic beta-glucosidase unravels the substrate aglycone specificity of a family 1 glycoside hydrolase

Human cytosolic beta-glucosidase (hCBG) is a xenobiotic-metabolizing enzyme that hydrolyses certain flavonoid glucosides, with specificity depending on the aglycone moiety, the type of sugar and the linkage between them. In this study, the substrate preference of this enzyme was investigated by mutational analysis, X-ray crystallography and homology modelling. The crystal structure of hCBG was solved by the molecular replacement method and refined at 2.7 A resolution. The main-chain fold of the enzyme belongs to the (beta/alpha)(8) barrel structure, which is common to family 1 glycoside hydrolases. The active site is located at the bottom of a pocket (about 16 A deep) formed by large surface loops, surrounding the C termini of the barrel of beta-strands. As for all the clan of GH-A enzymes, the two catalytic glutamate residues are located on strand 4 (the acid/base Glu165) and on strand 7 (the nucleophile Glu373). Although many features of hCBG were shown to be very similar to previously described enzymes from this family, crucial differences were observed in the surface loops surrounding the aglycone binding site, and these are likely to strongly influence the substrate specificity. The positioning of a substrate molecule (quercetin-4'-glucoside) by homology modelling revealed that hydrophobic interactions dominate the binding of the aglycone moiety. In particular, Val168, Trp345, Phe225, Phe179, Phe334 and Phe433 were identified as likely to be important in determining substrate specificity in hCBG, and site-directed mutagenesis supported a key role for some of these residues.     

On porcine pancreatic alpha-amylase action: kinetic evidence for the binding of two maltooligosaccharide molecules (maltose, maltotriose and o-nitrophenylmaltoside) by inhibition studies. Correlation with the five-subsite energy profile

Hydrolysis of small substrates (maltose, maltotriose and o-nitrophenylmaltoside) catalysed by porcine pancreatic alpha-amylase was studied from a kinetic viewpoint over a wide range of substrate concentrations. Non-linear double-reciprocal plots are obtained at high maltose, maltotriose and o-nitrophenylmaltoside concentrations indicating typical substrate inhibition. These results are consistent with the successive binding of two molecules of substrate per enzyme molecule with dissociation constants Ks1 and Ks2. The Hill plot, log [v/(V-v)] versus log [S], is clearly biphasic and allows the dissociation constants of the ES1 and ES2 complexes to be calculated. Maltose and maltotriose are inhibitors of the amylase-catalysed amylose and o-nitrophenylmaltoside hydrolysis. The inhibition is of the competitive type. The (apparent) inhibition constant Kiapp varies with the inhibitor concentration. These results are also consistent with the successive binding of at least two molecules of maltose or maltotriose per amylase molecule with the dissociation constants Ki1 and Ki2. These inhibition studies show that small substrates and large polymeric ones are hydrolysed at the same catalytic site(s). The values of the dissociation constants Ks1 and Ki1 of the maltose-amylase complexes are identical. According to the five-subsite energy profile previously determined, at low concentration, maltose (as substrate and as inhibitor) binds to the same two sites (4,5) or (3,4), maltotriose (as substrate and as inhibitor) and o-nitrophenyl-maltoside (as substrate) bind to the same three subsites (3,4,5). The dissociation constants Ks2 and Ki2 determined at high substrate and inhibitor concentration are consistent with the binding of the second ligand molecule at a single subsite. The binding mode of the second molecule of maltose (substrate) and o-nitrophenylmaltoside remains uncertain, very likely because of the inaccuracy due to simplifications in the calculations of the subsite binding energies. No binding site(s) outside the catalytic one has been taken into account in this model.     

P-Glycoprotein (P-gp) expressed in a confluent monolayer of hMDR1-MDCKII cells has more than one efflux pathway with cooperative binding sites

The multidrug resistance transporter P-glycoprotein (P-gp) effluxes a wide range of substrates and can be affected by a wide range of inhibitors or modulators. Many studies have presented classifications for these binding interactions, within either the context of equilibrium binding or the Michaelis-Menten enzyme analysis of the ATPase activity of P-gp. Our approach is to study P-gp transport and its inhibition using a physiologically relevant confluent monolayer of hMDR1-MDCKII cells. We measure the elementary rate constants for P-gp efflux of substrates and study inhibition using pairwise combinations with a different unlabeled substrate acting as the inhibitor. Our current kinetic model for P-gp has only a single binding site, because a previous study proved that the mass-action kinetics of efflux of a single substrate were not sensitive to whether there are one or more substrate-binding and efflux sites. In this study, using this one-site model, we found that, with "high" concentrations of either a substrate or an inhibitor, the elementary rate constants fitted independently for each of the substrates alone quantitatively predicted the efflux curves, simply applying the assumption that binding at the "one site" was competitive. On the other hand, at "low" concentrations of both the substrate and inhibitor, we found no inhibition of the substrate efflux, despite the fact that both the substrate and inhibitor were being well-effluxed. This was not an effect of excess "empty" P-gp molecules, because the competitive efflux model takes site occupancy into account. Rather, it is quantitative evidence that the substrate and inhibitor are being effluxed by multiple pathways within P-gp. Remarkably, increasing the substrate concentration above the "low" concentration, caused the inhibition to become competitive; i.e., the inhibitor became effective. These data and their analysis show that the binding of these substrates must be cooperative, either positive or negative.     

Substrate binding characteristics of the active site of spermidine dehydrogenase from Serratia marcescens.

The substrate specificity of spermidine dehydrogenase from Serratia marcescens was studied using many kinds of naturally occurring and synthetic polyamines. Diamines inhibited the enzyme competitively and their inhibitor constants tended to decrease with increasing methylene chain length in the diamines. All of the triamines and tetramines examined were active as substrates, and the amines containing a 4-aminobutylimino moiety (NH2(CH2)4NH-) in their structures were more active. N-Alkylputrescine was also oxidized by the enzyme. All of the amines containing a 4-aminobutylimino group were oxidized to form 1-pyrroline stoichiometrically as one of the products. Tetramines containing a 3-aminopropylimino group (NH2(CH2)3NH-) were oxidized to form 1,3-diaminopropane. However, in the case of an amine containing both 4-aminobutylimino and 3-aminopropylimino groups, the imino moiety of the former was preferentially oxidized by the enzyme. On the basis of the substrate specificity, the binding characteristics of the enzyme are discussed and a subsite model for the binding site is proposed.     

A reinvestigation of the substrate specificity of pig kidney diamine oxidase

1. The substrate specificity of pig kidney diamine oxidase was reinvestigated with a purer enzyme preparation than has previously been used for this purpose. 2. All substrates were extensively purified before use, and methods of preparation or sources are given, together with R(F) values. 3. The substrate specificity determined differed somewhat from that reported by previous workers and, in addition, the behaviour of several compounds not previously used as substrates is described. 4. A model for enzyme-substrate interaction embodying these observations is formulated. It is suggested that a negatively charged substrate-binding group is situated at 6.0-9.0 A from the oxidizing site. The binding and oxidizing sites are separated by a hydrophobic or methylene-binding site.     

Substrate inhibition in a human variant of hypoxanthine-guanine phosphoribosyltransferase

Hypoxanthine-guanine phosphoribosyltransferase from a young man with purine overproduction and decreased purine salvage in fibroblast cultures was found to have low activity at concentrations of purine substrates at which the enzyme from normal individuals showed near maximal activity. The low enzyme activity was not associated with changes in the values of the Km(app) and Vmax(app) for any of the enzyme substrates. However, the enzyme activity was susceptible to substrate inhibition by hypoxanthine and guanine. The values obtained for the true Km, true Vmax, and true Ki for hypoxanthine were 26 +/- 10 microM, 1761 +/- 382 microunits/mg of protein, and 80 +/- 20 microM, respectively. The pattern of the substrate inhibition, as seen on a plot of 1/v versus hypoxanthine concentration, was characteristic of that associated with the formation of a dead-end complex between the inhibitory substrate and an enzyme form with which it normally does not react. The nature of this enzyme form and that of the dead-end complex was determined from double inhibition experiments, which indicated that hypoxanthine interacted with an enzyme-PPi intermediate to form an enzyme-hypoxanthine-PPi dead-end complex. The trapping of the enzyme in this inactive form explains the low activity at high purine base concentrations. Further information as to the nature of the reaction mechanism was obtained from plots of the reciprocal of enzyme activity versus the reciprocal of PP-ribose-P concentration at different fixed hypoxanthine concentrations. A pattern characteristic of uncompetitive substrate inhibition was obtained. This is indicative of an ordered sequential binding of substrates on the enzyme; PP-ribose-P binding before hypoxanthine. Thus, the variant enzyme showed an ordered sequential reaction mechanism, with the inhibitory substrate forming a dead-end complex with an enzyme-PPi intermediate.     

Mechanistic studies with solubilized rat liver steroid 5 alpha-reductase: elucidation of the kinetic mechanism

A solubilized preparation of steroid 5 alpha-reductase (EC 1.3.1.30) from rat liver has been used in studies focused toward an understanding of the kinetic mechanism associated with enzyme catalysis. From the results of analyses with product and dead-end inhibitors, a preferentially ordered binding of substrates and release of products from the surface of the enzyme is proposed. The observations from these experiments were identical with those using the steroid 5 alpha-reductase activity associated with rat liver microsomes. The primary isotope effects on steady-state kinetic parameters when [4S-2H]NADPH was used also were consistent with an ordered kinetic mechanism. Normal isotope effects were observed for all three kinetic parameters (Vm/Km for both testosterone and NADPH and Vm) at all substrate concentrations used experimentally. Upon extrapolation to infinite concentration of testosterone, the isotope effect on Vm/Km for NADPH approached unity, indicating that the nicotinamide dinucleotide phosphate is the first substrate binding to and the second product released from the enzyme. The isotope effects on Vm/Km for testosterone at infinite concentration of cofactor and on Vm were 3.8 +/- 0.5 and 3.3 +/- 0.4, respectively. Data from the pH profiles of these three steady-state parameters and the inhibition constants (1/Ki) of competitive inhibitors versus both substrates indicate that the binding of nicotinamide dinucleotide phosphate involves coordination of its anionic 2'-phosphate to a protonated enzyme-associated base with an apparent pK near 8.0. From these results, relative limits have been placed on several of the internal rate constants used to describe the ordered mechanism of the rat liver steroid 5 alpha-reductase.     

Thermodynamic analysis of purified human placental alpha-L-fucosidase

The temperature dependence for the hydrolysis of both 4-methylumbelliferyl-α-l-fucoside and p-nitrophenyl-α-l-fucoside was determined for purified α-l-fucosidase (EC 3.2.1.51) from human placenta. The inhibition of the enzymatic reaction by l-fucose was also studied using the first of these two substrates at different temperatures. The thermodynamic parameters calculated from the pK_m were for the 4-methylumbelliferyl-conjugate ΔF = ?6.6 kcal/mol, ΔH = ?8.5 kcal/mol, and ΔS = ?6.3 e.u. and for the p-nitrophenylconjugate ΔF = ?5.6 kcal/mol, ΔH = ?12.2 kcal/mol, and ΔS = ?21.1 e.u. The thermodynamic parameters for l-fucose were ΔH = ?12.4 kcal/mol and ΔS = ?20.1 e.u. The lower exothermicity and negative entropy calculated for the 4-methylumbelliferyl substrate compared to the thermodynamic parameters calculated for the p-nitrophenyl substrate and l-fucose suggest the existence of a secondary hydrophobic binding site for the 4-methylumbelliferyl moiety on the enzyme. The difference in the enthalpy for both substrates is also reflected in a difference in activation energy, being 15.8 kcal/mol for the 4-methylumbelliferyl substrate and 20.7 kcal/mol for the p-nitrophenyl substrate. From these results it may be concluded that altered kinetic properties of the enzyme could be the result of the binding of the “aglycone” moiety of the fluorogenic substrate to the enzyme.     

Kinetics and energetics of ligand binding determined by microcalorimetry: insights into active site mobility in a psychrophilic alpha-amylase

A new microcalorimetric method for recording the kinetic parameters k(cat), K(m) and K(i) of alpha-amylases using polysaccharides and oligosaccharides as substrates is described. This method is based on the heat released by glycosidic bond hydrolysis. The method has been developed to study the active site properties of the cold-active alpha-amylase produced by an Antarctic psychrophilic bacterium in comparison with its closest structural homolog from pig pancreas. It is shown that the psychrophilic alpha-amylase is more active on large macromolecular substrates and that the higher rate constants k(cat) are gained at the expense of a lower affinity for the substrate. The active site is able to accommodate larger inhibitory complexes, resulting in a mixed-type inhibition of starch hydrolysis by maltose. A method for recording the binding enthalpies by isothermal titration calorimetry in a low-affinity system has been developed, allowing analysis of the energetics of weak ligand binding using the allosteric activator chloride. It is shown that the low affinity of the psychrophilic alpha-amylase for chloride is entropically driven. The high enthalpic and entropic contributions of activator binding suggest large structural fluctuations between the free and the bound states of the cold-active enzyme. The kinetic and thermodynamic data for the psychrophilic alpha-amylase indicate that the strictly conserved side-chains involved in substrate binding and catalysis possess an improved mobility, responsible for activity in the cold, and resulting from the disappearance of stabilizing interactions far from the active site.