Characterization of calf liver glucosidase I and its inhibition by basic sugar analogs

Glucosidase I, the enzyme catalyzing the first step of N-linked oligosaccharide processing, has been purified from calf liver crude membranes [H. Hettkamp, G. Legler, and E. Bause, (1984) Eur. J. Biochem. 142, 85-90]. Binding experiments with concanavalin A-Sepharose suggest that glucosidase I is a glycoprotein with high-mannose carbohydrate chain(s). The enzyme has a subunit molecular mass of approximately 83 kDa and specifically hydrolyzes the terminal alpha-1,2-linked glucose residue from the natural Glc3-Man9-GlcNAc2 oligosaccharide. Studies with a variety of substrates modified in the aglycon moiety suggest that the Glc2 branch rather than the more distant domains of the substrate molecule are important for binding and hydrolysis. Glucosidase I does not require metal ions for activity and is strongly inhibited by 1-deoxynojirimycin (dNM) and its N-alkyl derivatives. Ki values range from 0.07 microM for N-methyl-dNM to 1.0 microM for dNM, measured at the pH-optimum of enzyme activity. The pH dependence of inhibition indicates that the cationic form of the inhibitors is the active species. Comparison of the Ki for N-decanoyl-dNM (approximately 70 microM) with that of N-decyl-dNM (approximately 0.4 microM) suggests that electrostatic interactions at the catalytic site of the enzyme are important for inhibitor binding. 1-Deoxymannojirimycin, previously assumed to be a specific mannosidase inhibitor, as well as its N-methyl and N-5-carboxypentyl derivatives, inhibit glucosidase I with Ki values around 190, 17, and 100 microM, respectively. This apparent lack of specificity shows that in vivo experiments on N-glycoprotein processing as well as the interpretation of results with these mannosidase inhibitors may give misleading results when these compounds are used in the millimolar range.     

N-acetylglucosaminono-1,5-lactone oxime and the corresponding (phenylcarbamoyl)oxime. Novel and potent inhibitors of beta-N-acetylglucosaminidase

Using N-acetylglucosaminono-1,5-lactone (1) as the reference, the inhibitory activity of its (formal) derivatives N-acetylglucosaminono-1,5-lactone oxime (2) and N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)-oxime (3) was tested against beta-N-acetylglucosaminidase of different origins (animal, plant, fungus). Displaying inhibition constants of 0.45 microM and 0.62 microM, for the animal and plant enzyme, respectively, the simple oxime 2 was about equally potent as the parent lactone 1, and 50-400 times more efficient than two recently described new beta-N-acetylglucosaminidase inhibitors. The (phenylcarbamoyl)oxime 3 performed even better, particularly with the fungal enzyme (Ki = 40 nM), i.e. was about 350 times more potent than the lactone. In all cases competitive inhibition was observed with 4-nitrophenyl-beta-N-acetylglucosaminide as the substrate. With Ki/Km ratios up to 3300 for 2 and 13,600 for 3, the mode of action of these novel inhibitors is probably that of transition state mimicry. Suggestions are made for their use as a tool in biological research.     

Effects of 8-substituted adenosine 3',5'-monophosphate derivatives on high Km phosphodiesterase activity.

Most of twenty-one 8-substitued adenosine 3',5'-monophosphate derivatives were found to inhibit competitively the hydrolysis of adenosine 3'5'-monophosphate by partially purified high Km (Michaelis-Menten constant) phosphodiesterase from hog brain cortex, which had one active site at high concentration of adenosine 3',5'-monophosphate (0.3 to 4.0 mM). The Ki value for the 8-substituted alkylaminoadenosine 3'5'-monophosphate derivative was found to decrease with increasing unbranched carbon chain of the substituent, and a minimum value was obtained in the case of 8-octylaminoadenosine 3',5'-monophosphate. The Ki value, however, increased gradually as the substituent of derivative became longer than that of 8-octylminoadenosine 3'5'-monophosphate. The similar phenomenon was observed in the 8-substituted alkylthioadenosine 3',5'-monophosphate. The standard affinity for adenosine 3,5'-monophosphate of the high Km phosphodiesterase was 5.0 kcal/mol, which was calculated from Km. The standard affinity for 8-hexylthioadenosine 3',5'-monophosphate, which inhibited most strongly the enzyme activity, was 7.2 kcal/mol. The difference (2.2 kcal/736) between the standard affinity for adenosine 3',5'-monphosphate and that for 8-hexylthioadenosine 3',5'-monophosphate seems to be based on the partial affinity for the substituent (hexylthio group) of the active site on the enzyme or its neighborhood. A characteristic similar interrelation between substituent length of derivatives and their inhibitory effect on the enzyme activity was observed similarly in two different series of derivatives, 8-substituted alkylaminoadenosine 3',5'-monophosphate and alkylthioadenosine 3',5'-monophosphate. The results may indicate the characteristic structure of the active site of the enzyme or its neighborhood.     

Interaction of fluorescent adenine nucleotide derivatives with the ADP/ATP carrier in mitochondria. 1. Comparison of various 3'-O-ester adenine nucleotide derivatives

Fluorescent 3'-O-acyl-substituted adenine nucleotide (dimethylamino)naphthoyl and trinitrophenyl groups were studied for binding to the ADP/ATP carrier in mitochondria and submitochondrial particles. The changes in fluorescence intensity and emission maximum are for the most part similar to those observed in nonaqueous solvents. The (dimethylamino)naphthoyl derivatives from a largely quenched aqueous state have a shortwave shift up to 85 nm and increase up to 90-fold (1,5 derivative), whereas the little quenched naphthoyl derivatives show a fluorescence decrease and the weakly fluorescent trinitrophenyl derivative shows only a small increase on binding. All derivatives are good inhibitors (K1 = 1-10 microM) of nucleotide transport. The fluorescence titrations have an apparent K1/2 = 2-7 microM. The fluorescence of the 1,5-DAN nucleotide is fully suppressed by bongkrekate but only partially suppressed by carboxyatractylate. The fluorescence response is much stronger in submitochondrial particles than in mitochondria. Both facts suggest fluorescent binding to the "m" state of the carrier site at the inner face of the membrane. 1,5-DAN-AMP shows a slightly more efficient binding than DAN-ADP or DAN-ATP.     

Purification of the putative hormone-sensitive cyclic AMP phosphodiesterase from rat adipose tissue using a derivative of cilostamide as a novel affinity ligand

A "low Km" cAMP phosphodiesterase with properties of a peripheral membrane protein accounts for approximately 90% of total cAMP phosphodiesterase activity in particulate (100,000 X g) fractions from rat fat cells. Incubation of fat cells with insulin for 10 min increased particulate (but not soluble) cAMP phosphodiesterase activity, with a maximum increase (approximately 100%) at 1 nM insulin. Most of the increase in activity was retained after solubilization (with non-ionic detergent and NaBr) and partial purification (approximately 20-fold) on DEAE-Sephacel. The solubilized enzyme from adipose tissue was purified approximately 65,000-fold to apparent homogeneity (yield approximately 20%) by chromatography on DEAE-Sephacel and Sephadex G-200 and affinity chromatography on aminoethyl agarose conjugated with the N-(2-isothiocyanato)ethyl derivative of the phosphodiesterase inhibitor cilostamide (OPC 3689). A 63,800 +/- 200-Da polypeptide (accounting for greater than 90% of the protein eluted from the affinity column) was identified by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (with or without reduction). Enzyme activity was associated with the single protein band after electrophoresis under nondenaturing conditions. On gel permeation, Mr(app) was 100,000-110,000, suggesting that the holoenzyme is a dimer. A pI of 4.9-5.0 was estimated by isoelectric focusing. At 30 degrees C, the purified enzyme hydrolyzed both cAMP and cGMP with normal Michaelis-Menten kinetics; the pH optimum was 7.5. The Km(app) for cAMP was 0.38 microM and Vmax, 8.5 mumol/min/mg; for cGMP, Km(app) was 0.28 microM and Vmax, 2.0 mumol/min/mg. cGMP competitively inhibited cAMP hydrolysis with a Ki of approximately 0.15 microM. The enzyme was also inhibited by several OPC derivatives and "cardiotonic" drugs, but not by RO 20-1724. It was very sensitive to inhibition by agents which covalently modify protein sulfhydryls, but not by diisopropyl fluorophosphate. The activation by insulin and other findings indicate that the purified enzyme, which seems to belong to a subtype of low Km cAMP phosphodiesterases that is specifically and potently inhibited by cGMP, cilostamide, other OPC derivatives, and certain cardiotonic drugs, is likely to account for the hormone-sensitive particulate low Km cAMP phosphodiesterase activity of rat adipocytes.     

Evidence for two genetically distinct DNA primase activities specified by plasmids of the B and I incompatibility groups.

Soluble formate dehydrogenase from Methanobacterium formicicum was purified 71-fold with a yield of 35%. Purification was performed anaerobically in the presence of 10 mM sodium azide which stabilized the enzyme. The purified enzyme reduced, with formate, 50 mumol of methyl viologen per min per mg of protein and 8.2 mumol of coenzyme F420 per min per mg of protein. The apparent Km for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F420, was 10-fold greater (63 microM) than for coenzyme F420 (6 microM). The purified enzyme also reduced flavin mononucleotide (Km = 13 microM) and flavin adenine dinucleotide (Km = 25 microM) with formate, but did not reduce NAD+ or NADP+. The reduction of NADP+ with formate required formate dehydrogenase, coenzyme F420, and coenzyme F420:NADP+ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F420. The optimal reaction rate occurred at 55 degrees C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (Ki = 6 microM), azide (Ki = 39 microM), alpha,alpha-dipyridyl, and 1,10-phenanthroline. Denaturation of the purified formate dehydrogenase with sodium dodecyl sulfate under aerobic conditions revealed a fluorescent compound. Maximal excitation occurred at 385 nm, with minor peaks at 277 and 302 nm. Maximal fluorescence emission occurred at 455 nm.     

The K+-translocating Kdp-ATPase from Escherichia coli. Purification, enzymatic properties and production of complex- and subunit-specific antisera

The Kdp system from Escherichia coli is a derepressible high-affinity K+-uptake ATPase. Its membrane-bound ATPase activity was approximately 50 mumol g-1 min-1. The Kdp-ATPase complex was purified from everted vesicles by solubilization with the nonionic detergent Aminoxid WS 35 followed by DEAE-Sepharose CL-6B chromatography at pH 7.5 and pH 6.4 and gel filtration on Fractogel TSK HW-65. The overall yield of activity was 6.5% and the purity at least 90%. The isolated KdpABC complex had a high affinity for its substrates K+ (Km app. = 10 microM) and Mg2+-ATP (Km = 80 microM) and a narrow substrate specificity. The ATPase activity was inhibited by vanadate (Ki = 1.5 microM), fluorescein isothiocyanate (Ki = 3.5 microM), N,N'-dicyclohexylcarbodiimide (Ki = 60 microM) and N-ethylmaleimide (Ki = 0.1 mM). The purification protocol was likewise applicable to the isolation of a KdpA mutant ATPase which in contrast to the wild-type enzyme exhibited an increased Km value for K+ of 6 mM and a 10-fold lowered sensitivity for vanadate. Starting from the purified Kdp complex the single subunits were obtained by gel filtration on Bio-Gel P-100 in the presence of SDS. Both the native Kdp-ATPase and the SDS-denatured polypeptides were used to raise polyclonal antibodies. The specificity of the antisera was established by immunoblot analysis. In functional inhibition studies the anti-KdpABC and anti-KdpB sera impaired ATPase activity in the membrane-bound as well as in the purified state of the enzyme. In contrast, the anti-KdpC serum did not inhibit enzyme activity.     

Activation of rabbit muscle fructose 1,6-bisphosphatase by histidine and carnosine

Histidine and its derivatives increased rabbit muscle fructose 1,6-bisphosphatase activity at neutral pH with positive cooperativity. In the presence of histidine and carnosine the optimum pH shifted from pH 8.0 to 7.4. The cooperative response of the enzyme to AMP and fructose 1,6-bisphosphate was observed in the presence of the histidine derivatives. Of a number of divalent cations tested, only Zn2+ was found to be an effective inhibitor of enzyme activity at low concentrations. The kinetic data suggested that Zn2+ acted as inhibitor as well as activator for the enzyme activity; a high affinity binding site was associated with Ki of approximately 0.5 microM Zn2+ and a catalytic site was associated with Km of approximately 10 microM Zn2+. Rabbit muscle fructose 1,6-bisphosphatase bound 4 equivalents of Zn2+/mol, presumably 1 per subunit, in the absence of fructose 1,6-bisphosphate. Two equivalents of Zn2+/mol bound to the enzyme were readily removed by dialysis or gel filtration in the absence of a chelating agent. The other two equivalents of Zn2+/mol were removed by histidine and histidine derivatives of naturally occurring chelators with concomitant increase in activity.     

Inhibition of acetoacetyl-CoA synthetase from rat liver by fatty acyl-CoAs

The activity of acetoacetyl-CoA synthetase from rat liver was found to be negatively regulated by coenzyme A, fatty acyl-CoAs and acetoacetyl-CoA in vitro. With increasing concentrations of coenzyme A (substrate inhibition occurring at concentrations higher than 50 microM) the pH optimum shifted toward the acidic side (7.5-8.5 with 5 microM coenzyme A and 6.5-7.0 with 500 microM coenzyme A), in parallel with progressively decreasing enzyme activity. Fatty acyl-CoAs of various chain lengths dose-dependently inhibited acetoacetyl-CoA synthetase from rat liver, but much less effectively a similar enzyme from a bacterium, Zoogloea ramigera I-16-M. Palmitoyl-CoA, the most potent inhibitor of the rat liver enzyme, with an apparent Ki value of 9.8 microM, apparently inhibited the enzyme below its critical micellar concentration, not due to its detergent action. Acetoacetyl-CoA showed product inhibition with a Ki value of 15 microM. These results suggest a possible physiological regulation mechanism for this enzyme with respect to fatty acid biosynthesis.     

Pyruvate:NADP+ oxidoreductase from Euglena gracilis: the kinetic properties of the enzyme

The kinetic properties for the native forward reaction of pyruvate:NADP+ oxidoreductase from Euglena gracilis were determined. The substrate kinetics gave a pattern of a ping-pong mechanism involving a competitive substrate inhibition of CoA against pyruvate. The Km values for pyruvate, CoA, and NADP+ were estimated to be 27, 6.6, and 28 microM, respectively, and the Ki value of CoA against pyruvate was 28 microM. CO2 inhibited noncompetitively against pyruvate and NADP+, and uncompetitively against CoA. Acetyl-CoA showed a competitive inhibition with respect to pyruvate and an uncompetitive inhibition with respect to NADP+. NADPH inhibited competitively versus NADP+, noncompetitively versus CoA, and uncompetitively versus pyruvate. The kinetic behavior is consistent with a two-site ping-pong mechanism involving the substrate inhibition. From the kinetic mechanism, it is proposed that the enzyme has two catalytic sites linked by an intramolecular electron-transport chain. One of these is a thiamine pyrophosphate-containing catalytic site which reacts with pyruvate and CoA to form CO2 and acetyl-CoA, and the other site functions in the reduction of NADP+. In contrast, when methyl viologen was used as an artificial one-electron acceptor substituting for NADP+, the reaction gave a pattern characteristic of an octa uni ping-pong mechanism involving a competitive substrate inhibition of CoA against pyruvate.     

Relationship of the regulatory nucleotide site to the catalytic site of the sarcoplasmic reticulum Ca2+-ATPase

The purpose of this study was to probe the regulatory nucleotide site of the Ca2+-ATPase of sarcoplasmic reticulum and to study its relationship with the catalytic nucleotide site. Our approach was to use the nucleotide analogue 2'(3')-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-phosphate (TNP-AMP), which is known to bind the Ca2+-ATPase with high affinity and to undergo a manyfold increase in fluorescence upon enzyme phosphorylation with ATP in the presence of Ca2+. TNP-AMP was shown to bind the regulatory site in that it competitively inhibited (Ki = 0.6 microM) the secondary activation of turnover induced by millimolar ATP, thus providing a high affinity probe for the site. Observation of the high phosphoenzyme-dependent fluorescence upon monomerization of the enzyme without an increase in phosphoenzyme levels showed the regulatory site to be on the same subunit as the catalytic site and excluded an uncovering of "silent" nucleotide sites resulting from dissociation of enzyme subunits. Identical stoichiometric levels of [3H]TNP-AMP binding (4 nmol/mg of protein) to either the free enzyme or the enzyme phosphorylated with 250 microM ATP excluded models of two nucleotide sites per subunit. Finally, transient kinetic experiments in which TNP-AMP was found to block the ADP-induced burst of phosphoenzyme decomposition showed that TNP-AMP was bound to the phosphorylated catalytic site. We conclude that the regulatory nucleotide site is not a separate and distinct site on the Ca2+-ATPase but, rather, results from the nucleotide catalytic site following formation of the phosphorylated enzyme intermediate.     

Interactions of alpha-chymotrypsin and Carlsberg subtilisin with methyl N alpha-acetyl-2-(alkylthio)-L-tryptophanoates

Methyl N alpha-acetyl-2-(alkylthio)-L-tryptophanoates bearing different alkylthio groups were synthesized and employed as substrates for alpha-chymotrypsin and Carlsberg subtilisin in an attempt to investigate the properties of the hydrophobic pocket or cleft (S1 subsite) of the enzymes which accommodates the side-chain of the P1 amino acid residue of the substrates. The derivatives with ethylthio, 2-hydroxyethylthio, 2,3-dihydroxypropylthio, 2-aminoethylthio, carboxymethylthio, 2-carboxyethylthio, 1,2-dicarboxyethylthio, and 2-amino-2-carboxyethylthio (cysteinyl-S) groups were hydrolyzed by alpha-chymotrypsin but with kcat/Km values 4.6 to 15 times smaller than that of methyl N alpha-acetyl-L-tryptophanoate, due mainly to larger Km values. The glutathionyl derivative was only weakly bound to the enzyme. Analyses of the kinetic parameters suggested that the S1 pocket of alpha-chymotrypsin is rather more spacious than has been supposed and is able to interact flexibly with substrates so as to orient the scissile bond to the catalytic residues. On the other hand, none of the derivatives were hydrolyzed by Carlsberg subtilisin but they all inhibited the enzyme with Ki values which are generally smaller than the Km values for N alpha-acetyl-L-aromatic (modified aromatic) amino acid methyl esters. The S1 cleft of Carlsberg subtilisin interacts rather strongly with the derivatives but lacks the flexibility necessary for catalysis.     

Cyclic GMP-dependent stimulation of the membrane-bound insulin-sensitive cAMP phosphodiesterase from rat adipocytes

The insulin-sensitive cAMP phosphodiesterase (phosphodiesterase) in rat adipocytes is a membrane-bound low Km enzyme that can be recovered in a crude microsomal fraction (Fraction P-2). The action of this enzyme to hydrolyze cAMP is known to be inhibited by cGMP; nevertheless, it was found in our present study that under selected conditions, the enzyme can also be stimulated by cGMP as well as some other nucleotide derivatives. The maximum cGMP-dependent stimulation was observed when the enzyme in Fraction P-2 was incubated with 10 microM cGMP for 5-20 min at 37 degrees C in the presence of Mg2+, washed, and then assayed in the absence of added cGMP. The level of this stimulation was close to, but less than, that achieved by insulin in intact cells. The actions of the cGMP- and insulin-stimulated enzymes to hydrolyze labeled cAMP were inhibited in an identical manner by cilostamide (Ki = 0.10 microM), griseolic acid (Ki = 0.19 microM), unlabeled cAMP (Km = 0.20 microM), and cGMP (Ki = 0.16 microM), all added to the assay system. Also, the basal, insulin-stimulated, and cGMP-activated enzymes were identically inhibited by a polyclonal antibody raised against a purified membrane-bound low Km phosphodiesterase from bovine adipose tissue. When the same antibody was used for the Western blot analysis of Fraction P-2, it immunoreacted with a single band of protein (165 kDa). These observations indicate that the insulin-sensitive phosphodiesterase in rat adipocytes can be stimulated with 10 microM cGMP and that this stimulation is detectable only after the nucleotide has been eliminated since the enzyme would be strongly inhibited by the nucleotide if the latter exists in the assay system. It is proposed that the insulin-sensitive phosphodiesterase, which is often referred to as a Type IV enzyme, is functionally similar to the Type II enzymes that are known to be stimulated by a low concentration of cGMP and inhibited by higher concentrations of the same nucleotide.     

Interaction of pseudomonic acid A with Escherichia coli B isoleucyl-tRNA synthetase.

Sodium pseudomonate was shown to be a powerful competitive inhibitor of Escherichia coli B isoleucyl-tRNA synthetase (Ile-tRNA synthetase). The antibiotic competitively inhibits (Ki 6 nM; cf. Km 6.3 microM), with respect top isoleucine, the formation of the enzyme . Ile approximately AMP complex as measured by the pyrophosphate-exchange reaction, and has no effect on the transfer of [14C]isoleucine from the enzyme . [14C]Ile approximately AMP complex to tRNAIle. The inhibitory constant for the pyrophosphate-exchange reaction was of the same order as that determined for the inhibition of the overall aminoacylation reaction (Ki 2.5 nM; cf. Km 11.1 microM). Sodium [9'-3H]pseudomonate forms a stable complex with Ile-tRNA synthetase. Gel-filtration and gel-electrophoresis studies showed that the antibiotic is only fully released from the complex by 5 M-urea treatment or boiling in 0.1% sodium dodecyl sulphate. The molar binding ratio of sodium [9'-3H]pseudomonate to Ile-tRNA synthetase was found to be 0.85:1 by equilibrium dialysis. Aminoacylation of yeast tRNAIle by rat liver Ile-tRNA synthetase was also competitively inhibited with respect to isoleucine, Ki 20 microM (cf. Km 5.4 microM). The Km values for the rat liver and E. coli B enzymes were of the same order, but the Ki for the rat liver enzyme was 8000 times the Ki for the E. coli B enzyme. This presumably explains the low toxicity of the antibiotic in mammals.     

Effect of GTP analogues on purified soluble guanylate cyclase

In our studies with purified soluble guanylate cyclase from rat lung, we have tested a number of guanosine 5'-triphosphate (GTP) analogues as substrates and inhibitors, 5'-Guanylylimidodiphosphate (GMP-P(NH)P), guanylyl (beta, gamma-methylene) diphosphate (GMP-P(CH2)P), and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) were found to be substrates for guanylate cyclase. GTP gamma S supported cyclic GMP formation at 20 or 75% of the rate seen with Mn2+-GTP and Mg2+-GTP, respectively. GMP-P(NH)P and GMP P(CH2)P supported cyclic GMP formation at 10-20% of the GTP rate with either cation cofactor. These analogues were found to have multiple Km values; one Km value was similar to GTP (150 microM with Mg2+, 20-70 microM with Mn2+), but an additional high affinity catalytic site (3 microM) was also observed. Guanosine tetraphosphate (Ki = 10 microM), adenosine triphosphate (Ki = 9 microM) and the 2'3'-dialdehyde derivative of GTP (dial GTP) (Ki = 1 microM) were not good substrates for the enzyme; however, they were potent competitive inhibitors. These GTP analogues will be useful tools for the study of GTP binding sites on guanylate cyclase and they may also help elucidate the effects of free radicals and other agents on guanylate cyclase regulation.     

Mouse protoporphyrinogen oxidase. Kinetic parameters and demonstration of inhibition by bilirubin.

The penultimate step of haem biosynthesis, the oxidation of protoporphyrinogen to protoporphyrin, was examined with purified murine hepatic protoporphyrinogen oxidase (EC 1.3.3.4) in detergent solution. The kinetic parameters for the two-substrate (protoporphyrinogen and oxygen) reaction were determined. The limiting Km for protoporphyrinogen when oxygen is saturating is 6.6 microM, whereas the Km for oxygen with saturating concentrations of protoporphyrinogen is 125 microM. The kcat. for the overall reaction is 447 h-1. The ratio of kcat. to the Km for protoporphyrinogen is approx. 20-fold greater than the kcat./Km,O2 ratio. The ratio of protoporphyrin formed to dioxygen consumed is 1:3. Ubiquinone-6, ubiquinone-10 and dicoumarol stimulate protoporphyrinogen oxidase activity at low concentrations (less than 15 microM), whereas coenzyme Q0 and menadione show no activation at these concentrations. Above 30 microM, all five quinones inhibit the enzyme activity. FAD does not significantly affect the activity of the enzyme. Bilirubin, a product of haem catabolism, is shown to be a competitive inhibitor of the penultimate enzyme of the haem-biosynthetic pathway, protoporphyrinogen oxidase, with a calculated Ki of 25 microM. The terminal enzyme of haem-biosynthetic pathway, namely ferrochelatase, is not inhibited by bilirubin at concentrations over double the Ki value for the oxidase. In contrast with other enzymic systems, the toxicity of bilirubin is not reversed by binding to albumin.     

Isolation of a cytosolic beta-glucosidase from calf liver and characterization of its active site with alkyl glucosides and basic glycosyl derivatives

A beta-glucosidase/beta-galactosidase with Mr 52,500 was isolated from calf liver cytosol by a four-step procedure incorporating affinity chromatography on N-(9-carboxynonyl)-deoxynojirimycin-AH-Sepharose. Its pH optimum was at 5.8 with half-maximal activity at pH 3.5 and 8.6. Affinity for gluco compounds expressed by Km or Ki of substrates and inhibitors was 2- to 10-fold higher than for the corresponding galacto compounds. Alkyl glucosides were hydrolyzed with lower Vmax than p-nitrophenyl and 4-methylumbelliferyl glucosides, but due to their higher affinity the alkyl glucosides displayed values for kcat/Km of the same magnitude of the aryl glucosides when the alkyl chains were longer than octyl. Glucosylsphingosine was bound with Ki (= Km) 2.2 microM and hydrolyzed with a Vmax that was 50-fold lower than the Vmax for 4-methylumbelliferyl beta-glucoside. The product sphingosine was inhibitory with Ki 0.30 microM. A systematic study with alkyl glucosides and glucosylamines defined the aglycon site as a narrow, strongly hydrophobic cleft able to accommodate up to 10 methylene groups. Each CH2 group contributed 3.1 kJ/mol to the standard free energy of binding. The inhibition by gluco- and galactosylamine and by 1-deoxynojirimycin and its D-galacto analog was approximately 200-fold better than by corresponding nonbasic compounds. pH dependence of the inhibition and comparison with permanently cationic glycosyl derivatives showed that the nonprotonated form was the inhibiting species. This feature puts the cytosolic beta-glucosidase in the large class of glycoside hydrolases which strongly bind basic glycosyl derivatives by their protonation at the active site and formation of a shielded ion pair with the carboxylate of an aspartic or glutamic side chain.     

Inhibition of adenylate cyclase by the 2',3'-dialdehyde of adenosine triphosphate

The periodate-oxidized analog of ATP, 2',3'-dialATP, competitively inhibited bovine brain and rat liver adenylate cyclase. The apparent Ki for inhibition of brain adenylate cyclase by 2',3'-dialATP was 196 microM in the presence of Mg2+ and 37 microM in the presence of Mn2+. The Ki values for inhibition of rat liver adenylate cyclase by 2',3'-dialATP were 48 and 30 microM in the presence of Mg2+; and Mn2+, respectively. Adenylate cyclase activity was irreversibly inactivated by 2'3'-dialATP in the presence of NaCNBH3 and the kinetics for loss in enzyme activity were pseudo-first order. Both ATP and Tris protected adenylate cyclase from irreversible inhibition by 2',3'-dialATP and NaCNBH3. It is proposed that 2',3'-dialATP forms a Schiff's base with an amino group at the active site of the enzyme and that Na-CNBH3 reduction of this Schiff's base causes irreversible modification of the catalytic subunit. The Km for 2',3'-dialATP inactivation, the maximal rate constant of inactivation, and protection of the enzyme by ATP were not affected by the presence or absence of free Mg2+. These data indicate that a divalent cation is not required for binding of 2',3'-dialATP to the active site of adenylate cyclase.     

MR 20492 and MR 20494: two indolizinone derivatives that strongly inhibit human aromatase.

In this study, we describe the synthesis of a new family of indolizinone derivatives designed to fit an extrahydrophobic pocket within the active site of aromatase and to strongly inhibit human aromatase. This could help improve the specificity of the inhibitors. Equine aromatase, very well characterized biochemically, is used as a comparative model. Indeed, in a previous comparison between both human and equine aromatases, we described the importance of the interaction between the inhibitor and this pocket for the indane derivative MR 20814. MR 20492 and MR 20494 are more potent inhibitors of human aromatase (Ki/Km: 1.0+/-0.3 and 0.5+/-0.3, respectively). The Ki/Km for MR 20494 is slightly higher than that obtained for fadrozole (0.1+/-0.0) and Ki/Km for both indolizinone derivatives are lower than those obtained for 4-hydroxyandrostenedione (1.9+/-0.8) and MR 20814 (8.1+/-.7). These new compounds are not enzyme inactivators. Moreover, as indicated by the higher Ki/Km values obtained with equine enzyme (9.0+/-0.6 and 6.1+/-1.6 for MR 20492 and MR 20494, respectively), both human and equine aromatase active sites appear to be structurally different. Difference absorption spectra study (350-500 nm) revealed that MR20492 and MR20494 were characterized by a combination of type-I and -II spectra with both enzymes. This result could be due to the isomerization of the molecule in polar solvent (Z and E forms). The evaluation of these new molecules, as well as 4-hydroxyandrostenedione and fadrozole, on aromatase activity in transfected 293 cell cultures evidenced a strong inhibition (IC50: 0.20+/-0.03 microM, 0.20+/-0.02 microM and 0.50+/-0.40 microM for MR 20494, fadrozole and 4-OHA, respectively) except for MR 20492 (3.9+/-0.9 microM) and MR 20814 (10.5+/-0.6 microM). These results proved that these molecules formed part of a promising family of potent inhibitors and that they penetrate 293 cells, without evidencing any cytotoxicity in Hela cells with MTT assay. This is thus encouraging for the development of new drugs for the treatment of estrogen-dependent cancers, these molecules also constitute new tools for understanding the aromatase active site.     

Human acid beta-glucosidase: use of inhibitors, alternative substrates and amphiphiles to investigate the properties of the normal and Gaucher disease active sites

Comparative studies with lipoidal inhibitors and alternative substrates were conducted to investigate the properties of the active site of human acid beta-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) from normal placenta and spleens of Type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients. With the normal enzyme, the inhibitory potencies of series of alkyl(Cn; n = 0-18)amines, alkyl beta-glucosides and alkyl-1-deoxynojirimycins were a biphasic function of increasing chain length: i.e., large decreases in Ki,app or IC50 were found only with n greater than 4 and limiting values were approached with n = 12-14. This biphasic function of alkyl chain length was observed in the presence or absence of detergents and/or negatively charged lipids. In the presence of Triton X-100 concentrations greater than the critical micellar concentration, the relative (to deoxynojirimycin) inhibitory potencies of the N-Cn-deoxynojirimycins (n greater than 4) were decreased about 3-5-fold, due to an energy requirement to extract the inhibitors from Triton X-100 micelles. The Ki,app or IC50 of N-hexylglucosylsphingosine was inversely related to the Triton X-100 concentration and was not affected by the presence of 'co-glucosidase'. The mutual exclusion of glucon, N-Cn-deoxynojirimycin and sphingosine derivatives from the normal enzyme suggested a shared region for binding in the active site. Increasing the fatty-acid acyl chain length of glucosyl ceramide from 1 to 24 carbons had minor effects on Km,app ( = Kis,app) (8-40 microM), but increased Vmax,app up to 13-fold. With the AJGD enzyme, the inhibitor and alternative substrate findings were similar to those with the normal enzyme, except that Kis,app(AJGD)/Kis,app(normal) = 4 to 11 for the Cn-glycons and sphingosine derivatives. These results indicated that (1) the Ki,app or Km,app values for amphiphilic inhibitors or substrates reflect a balance of binding energies for two hydrophobic subsites within the enzyme's active site and Triton X-100 micelles and (2) the abnormal properties of the AJGD enzyme result from an amino-acid alteration(s) within or near a hydrophilic region which is shared by the glycon-binding site and the two hydrophobic sites of the active site.