Oxidation and reduction of 4-hydroxyalkenals catalyzed by isozymes of human alcohol dehydrogenase

4-Hydroxyalkenals, natural cytotoxic products of lipid peroxidation, are substrates for human alcohol dehydrogenases (ADH). Class I and II ADHs reduce aliphatic 4-hydroxyalkenals with chain lengths of from 5 to 15 carbons at pH 7 with kcat and Km values comparable to simple aliphatic aldehydes of the same chain length. Class II is particularly effective in the reduction with kcat values as high as 3300 min-1 for 4-hydroxyundecenal. Class III ADH is essentially inactive toward all of these substrates. The class I and II isozymes also catalyze the oxidation of the 4-hydroxy group at pH 10. However, during the reaction, an NAD(+)-dependent irreversible partial inactivation of the alpha beta 1 isozyme is observed which is attributed, with the aid of computer graphics modeling, to selective modification of the alpha subunit. Both ethanol and 1,10-phenanthroline, known to compete with conventional substrates, instantaneously, reversibly, and competitively inhibit 4-hydroxyalkenal reduction and oxidation, indicating that 4-hydroxyalkenals bind at the same site as do conventional substates. The fact that the class II enzyme pi pi-ADH so far is found only in the liver and that the 4-hydroxyalkenals are the best substrates known for this isozyme suggest that it may play a significant role in cellular defenses in the conversion of the cytotoxic aldehydes to the less reactive alcohols.     

Purification and characterization of a new alcohol dehydrogenase from human stomach

Starch gel electrophoresis of homogenates from human stomach mucosa resolves three alcohol dehydrogenase (ADH) forms: the anodic chi-ADH (class III), the cathodic gamma-ADH (class I), and a new form of slow cathodic mobility that has not been previously characterized. In this work, we describe the purification in three chromatographic steps and the physical and kinetic characterization of this new human alcohol dehydrogenase, which we have named sigma-ADH. The enzyme exhibits the general physicochemical features (Mr, zinc content, subunit Mr, cofactor preference) of all mammalian alcohol dehydrogenases. The kinetic studies show a high Km value (41 mM) and a high kcat value (280 min-1) for ethanol at pH 7.5. The Km decreases as the alcohol increases its chain length. The aldehydes are better substrates than the corresponding alcohols, with m-nitrobenzaldehyde being the best substrate examined. sigma-ADH is strongly inhibited by 4-methylpyrazole, but with a Ki (10 microM) still higher than that for a class I isoenzyme. These properties suggest that sigma-ADH is a class II isoenzyme, different from pi-ADH and similar to that previously described by us in rat stomach. At the high ethanol concentrations in stomach after drinking, sigma-ADH is probably the ADH form with the largest contribution to human gastric ethanol metabolism.     

Human class I alcohol dehydrogenases catalyze the interconversion of alcohols and aldehydes in the metabolism of dopamine

The class I human liver alcohol dehydrogenases (ADHs) catalyze the interconversion of the intermediary alcohols and aldehydes of dopamine metabolism in vitro, whereas those of the class II and class III do not. The individual, homogeneous class I isozymes oxidize (3,4-dihydroxyphenyl)ethanol and (4-hydroxy-3-methoxyphenyl)ethanol (HMPE) and ethanol with kcat/Km values in the range from 16 to 240 mM-1 min-1 and from 16 to 66 mM-1 min-1, respectively. They reduce the corresponding dopamine aldehydes (3,4-dihydroxyphenyl)acetaldehyde and (4-hydroxy-3-methoxyphenyl)acetaldehyde (HMPAL) with kcat/Km values varying from 7800 to 190,000 mM-1 min-1, considerably more efficient than the reduction of acetaldehyde with kcat/Km values from 780 to 4900 mM-1 min-1. For beta 1 gamma 2 ADH, ethanol competes with HMPE oxidation with a Ki of 23 microM. In addition, 1,10-phenanthroline inhibits HMPE oxidation and HMPAL reduction with Ki values of 20 microM and 12 microM, respectively, both quite similar to that for ethanol, Ki = 22 microM. Thus, both ethanol/acetaldehyde and the dopamine intermediates compete for the same site of ADH, a basis for the ethanol-induced in vivo alterations of dopamine metabolism.     

Kinetic properties of human liver alcohol dehydrogenase: oxidation of alcohols by class I isoenzymes

Class I isoenzymes of alcohol dehydrogenase (ADH) were isolated by chromatography of human liver homogenates on DEAE-cellulose, 4-[3-[N-(6-aminocaproyl)-amino]propyl]pyrazole--Sepharose and CM-cellulose. Eight isoenzymes of different subunit composition (alpha gamma 2, gamma 2 gamma 2, alpha gamma 1, alpha beta 1, beta 1 gamma 2, gamma 1 gamma 1, beta 1 gamma 1, and beta 1 beta 1) were purified, and their activities were measured at pH 10.0 by using ethanol, ethylene glycol, methanol, benzyl alcohol, octanol, cyclohexanol, and 16-hydroxyhexadecanoic acid as substrates. Values of Km and kcat for all the isoenzymes, except beta 1 beta 1-ADH, were similar for the oxidation of ethanol but varied markedly for other alcohols. The kcat values for beta 1 beta 1-ADH were invariant (approximately 10 min-1) and much lower (5-15-fold) than those for any other class I isoenzyme studied. Km values for methanol and ethylene glycol were from 5- to 100-fold greater than those for ethanol, depending on the isoenzyme, while those for benzyl alcohol, octanol, and 16-hydroxyhexadecanoic acid were usually 100-1000-fold lower than those for ethanol. The homodimer beta 1 beta 1 had the lowest kcat/Km value for all alcohols studied except methanol and ethylene glycol; kcat values were relatively constant for all isoenzymes acting on all alcohols, and, hence, specificity was manifested principally in the value of Km. Values of Km and kcat/Km revealed for all enzymes examined that the short chain alcohols are the poorest while alcohols with bulky substituents are much better substrates. The experimental values of the kinetic parameters for heterodimers deviate from the calculated average of those of their parent homodimers and, hence, cannot be predicted from the behavior of the latter. Thus, the specificities of both the hetero- and homodimeric isoenzymes of ADH toward a given substrate are characteristics of each. Ethanol proved to be one of the "poorest" substrates examined for all class I isoenzymes which are the predominant forms of the human enzyme. On the basis of kinetic criteria, none of the isoenzymes of class I studied oxidized ethanol in a manner that would indicate an enzymatic preference for that alcohol.     

Purification and molecular properties of a Class II alcohol dehydrogenase (ADH-C2) from horse liver

An alcohol dehydrogenase (ADH) from horse liver was purified by ion-exchange and affinity chromatography. The enzyme (designated ADH-C2), is a dimer with a similar subunit size (47,300 mol. wt), as determined by SDS-polyacrylamide gel electrophoresis, to other mammalian ADHs. Zinc analyses and 1,10 phenanthroline inhibition studies indicated that each subunit contained 2 g atoms of zinc, with at least one involved catalytically. The enzyme exhibited similar kinetic properties to human pi-ADH and mouse ADH-C2, previously classified as class II ADHs [Vallee and Bazzone (1983) Isozymes, Vol. 8, pp. 219-244; Algar et al. (1983) Eur. J. Biochem. 137, 139-147] but differed in most respects from the extensively investigated horse Class I ADHs; EE, ES and SS. Horse ADH-C2 exhibited a Km value for ethanol of 42 mM and a broad substrate specificity, with Km values decreasing dramatically with an increase in chain length. The enzyme was much less sensitive to pyrazole inhibition (by at least 3 orders of magnitude) as compared with the Class I ADHs.     

Replacement of lysine 269 by arginine in Escherichia coli tryptophan indole-lyase affects the formation and breakdown of quinonoid complexes

Lysine 269 in Escherichia coli tryptophan indole-lyase (tryptophanase) has been changed to arginine by site-directed mutagenesis. The resultant K269R mutant enzyme exhibits kcat values about 10% those of the wild-type enzyme with S-(o-nitrophenyl)-L-cysteine, L-tryptophan, and S-benzyl-L-cysteine, while kcat/Km values are reduced to 2% or less. The pH profile of kcat/Km for S-benzyl-L-cysteine for the mutant enzyme exhibits two pK alpha values which are too close to separate, with an average value of 7.6, while the wild-type enzyme exhibits pK alpha values of 6.0 and 7.8. The pK alpha for the interconversion of the 335 and 412 nm forms of the K269R enzyme is 8.3, while the wild-type enzyme exhibits a pK alpha of 7.4. Steady-state kinetic isotope effects on the reaction of [alpha-2H]S-benzyl-L-cysteine with the K269R mutant enzyme (Dkcat = 2.0; D(kcat/Km) = 3.9) are larger than those of the wild-type enzyme (Dkcat = 1.4; D(kcat/Km) = 2.9). Rapid scanning stopped-flow kinetic studies demonstrate that the K269R mutant enzyme does not accumulate quinonoid intermediates with L-alanine, L-tryptophan, or S-methyl-L-cysteine, but does form quinonoid absorption peaks in complexes with S-benzyl-L-cysteine and oxidolyl-L-alanine, whereas wild-type enzyme forms prominent quinonoid bands with all these amino acids. Single wavelength stopped-flow kinetic studies demonstrate that the alpha-deprotonation of S-benzyl-L-cysteine is 6-fold slower in the K269R mutant enzyme, while the intrinsic deuterium kinetic isotope effect is less for the K269R enzyme (Dk = 4.2) than for the wild-type (Dk = 7.9). The decay of the K269R quinonoid intermediate in the presence of benzimidazole is 7.1-fold slower than that of the wild-type enzyme. These results demonstrate that Lys-269 plays a significant role in the conformational changes or electrostatic effects obligatory to the formation and decomposition of the quinonoid intermediate, although it is not an essential basic residue.     

3 beta-Hydroxy-5 beta-steroid dehydrogenase activity of human liver alcohol dehydrogenase is specific to gamma-subunits

Human liver alcohol dehydrogenase [alcohol:NAD+ oxidoreductase, EC 1.1.1.1 (ADH)] catalyzes the stereospecific oxidation of different 3 beta-hydroxy-5 beta-steroids with ranges of Km from 46 to 320 microM and values of kcat from 7.0 to 72 min-1, pH 8.5. Only the class I isozymes containing gamma-subunits, gamma 1 gamma 1, alpha gamma 1, beta 1 gamma 1, gamma 2 gamma 2, and beta 1 gamma 2, catalyze oxidation of these steroids with kcat/Km ratios 4-10-fold greater than those for ethanol. In marked contrast, class I alpha alpha, alpha beta 1, and beta 1 beta 1, class II, and class III isozymes do not oxidize 3 beta-hydroxy-5 beta-steroids though they readily oxidize ethanol. 1,10-Phenanthroline and 4-methylpyrazole competitively inhibit both alcohol dehydrogenase catalyzed ethanol and 3 beta-hydroxy-5 beta-steroid oxidation demonstrating that the catalysis of both types of substrates occurs at the same active site. The gamma-subunit-catalyzed oxidation of 3 beta-hydroxy-5 beta-steroids is the most specific catalytic function described thus far for any human liver alcohol dehydrogenase isozyme: there is no other isozyme that catalyzes this reaction. Testosterone, an allosteric inhibitor of ethanol oxidation specific for gamma-subunit-containing human liver ADH isozymes [M?rdh, G., Falchuk, K. H., Auld, D. S., & Vallee, B. L. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2836-2840], also noncompetitively inhibits gamma-subunit-catalyzed sterol oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial reversal of the acetaldehyde and butyraldehyde oxidation reactions catalysed by aldehyde dehydrogenases from sheep liver.

In the presence of acetic anhydride or butyric anhydride, liver aldehyde dehydrogenases catalyse the oxidation of NADH at pH 7.0 and 25 degrees C. The maximum velocities and Michaelis constants for NADH at saturating anhydride concentrations are independent of which anhydride is used, the values being V'max. = 12 min-1 and Km for NADH = 9 micrometer for the mitochondrial enzyme and V'max = 25 min-1 and Km for NADH = 20 micrometer for the cytoplasmic enzyme. Substitution of [4A-2H]NADH for NADH resulted in 2-fold and 4-fold decreases in rate for the mitochondrial and cytoplasmic enzymes respectively.     

Proteins of the kidney microvillar membrane. Purification and properties of carboxypeptidase P from pig kidneys.

Carboxypeptidase P has been purified by immunoaffinity chromatography from pig kidneys. A single-step assay with Z-Pro-Met (where Z represents benzyloxycarbonyl) as substrate was used, methionine being determined by using L-amino acid oxidase and horseradish peroxidase. The enzyme constitutes about 1.5% of the kidney microvillar proteins. Triton X-100-solubilized and papain-released forms of the enzyme were isolated. The former had an apparent subunit Mr of 135 000, and the latter form contained two polypeptide chains of Mr 128 000 and 95 000. The undenatured forms were dimeric proteins. In common with other microvillar hydrolases, carboxypeptidase P was a glycoprotein and each subunit contained one Zn atom. MnCl2 (1 mM) in the assay was necessary for maximum activity; in its absence, 0.5 mM-ZnSO4 produced a limited activation, but was inhibitory at higher concentrations. The Km for Z-Pro-Met, in the presence of MnCl2, was 4.1 mM, and the kcat. for freshly prepared enzyme was 1230 min-1. The enzyme lost activity during storage at -20 degrees C. In a limited survey of peptides, hydrolysis was observed only with substrates containing a proline, alanine or glycine residue in the P1 position, and these included angiotensins II and III. The best substrate in this series was Val-Ala-Ala-Phe.     

Novel activity of angiotensin-converting enzyme. Hydrolysis of cholecystokinin and gastrin analogues with release of the amidated C-terminal dipeptide.

ACE (angiotensin-converting enzyme; peptidyl dipeptidase A; EC 3.4.15.1), cleaves C-terminal dipeptides from active peptides containing a free C-terminus. We investigated the hydrolysis of cholecystokinin-8 [CCK-8; Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] and of various gastrin analogues by purified rabbit lung ACE. Although these peptides are amidated at their C-terminal end, they were metabolized by ACE to several peptide fragments. These fragments were analysed by h.p.l.c., isolated and identified by comparison with synthetic fragments, and by amino acid analysis. The initial and major site of hydrolysis was the penultimate peptide bond, which generated a major product, the C-terminal amidated dipeptide Asp-Phe-NH2. As a secondary cleavage, ACE subsequently released di- or tri-peptides from the C-terminal end of the remaining N-terminal fragments. The cleavage of CCK-8 and gastrin analogues was inhibited by ACE inhibitors (Captopril and EDTA), but not by other enzyme inhibitors (phosphoramidon, thiorphan, bestatin etc.). Hydrolysis of [Leu15]gastrin-(14-17)-peptide [Boc (t-butoxycarbonyl)-Trp-Leu-Asp-Phe-NH2] in the presence of ACE was found to be dependent on the chloride-ion concentration. Km values for the hydrolysis of CCK-8, [Leu15]gastrin-(11-17)-peptide and Boc-[Leu15]gastrin-(14-17)-peptide at an NaCl concentration of 300 mM were respectively 115, 420 and 3280 microM, and the catalytic constants were about 33, 115 and 885 min-1. The kcat/Km for the reactions at 37 degrees C was approx. 0.28 microM-1.min-1, which is approx. 35 times less than that reported for the cleavage of angiotensin I. These results suggest that ACE might be involved in the metabolism in vivo of CCK and gastrin short fragments.     

Human acid beta-glucosidase. Use of conduritol B epoxide derivatives to investigate the catalytically active normal and Gaucher disease enzymes

Human acid beta-glucosidase (glucosylceramidase; EC 3.2.1.45) cleaves the glycosidic bonds of glucosyl ceramide and synthetic beta-glucosides. Conduritol B epoxide (CBE) and its brominated derivative are mechanism-based inhibitors which bind covalently to the catalytic site of acid beta-glucosidase. Procedures using brominetritiated CBE and monospecific anti-human placental acid beta-glucosidase IgG were developed to determine the molar concentrations of functional acid beta-glucosidase catalytic sites in pure placental enzyme preparations from normal sources; kcat values then were calculated from Vmax = [Et]kcat using glucosyl ceramide substrates with dodecanoyl (2135 +/- 45 min-1) and hexanoyl (3200 +/- 410 min-1) fatty acid acyl chains and 4-alkyl-umbelliferyl beta-glucoside substrates with methyl (2235 +/- 197 min-1), heptyl (1972 +/- 152 min-1), nonyl (2220 +/- 247 min-1), and undecyl (773 +/- 44 min-1) alkyl chains. The respective kcat values for acid beta-glucosidase in a crude normal splenic preparation were about 60% of these values. In comparison, the kcat values of the mutant splenic acid beta-glucosidase from two Type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients were about 1.5-3-fold decreased and had Km values for each substrate which were similar to those for the normal acid beta-glucosidase. The interaction of the normal and Type 1 AJGD enzymes with CBE in a 1:1 stoichiometry conformed to a model with reversible EI complexes formed prior to covalent inactivation. With CBE, the equal kmax values (maximal rate of inactivation) for the normal (0.051 +/- 0.009 min-1) and Type 1 AJGD (0.058 +/- 0.016 min-1) enzymes were consistent with the minor differences in kcat. In contrast, the Ki value (dissociation constant) (839 +/- 64 microM) for the Type 1 AJGD enzymes was about 5 times the normal Ki value (166 +/- 57 microM). These results indicated that the catalytically active Type 1 AJGD acid beta-glucosidase had nearly normal hydrolytic capacity and suggested an amino acid substitution in or near the acid beta-glucosidase active site leading to an in vivo instability of the mutant enzymatic activity.     

Characterization of a low redox potential laccase from the basidiomycete C30.

A new exocellular laccase was purified from the basidiomycete C30. LAC2 is an acidic protein (pI = 3.2) preferentially produced upon a combined induction by copper and p-hydroxybenzoate. The spectroscopic signature (UV/visible and EPR) of this isoform is typical of multicopper oxidases, but its enzymatic and physico-chemical properties proved to be markedly different from those of LAC1, the constitutive laccase previously purified from the same organism. In particular, the LAC2 kcat values observed for the oxidation of the substrates syringaldazine (kcat = 65 600 min-1), ABTS (2,2-azino-bis-[3-ethylthiazoline-6-sulfonate] (kcat = 41 000 min-1) and guaiacol (kcat = 75 680 min-1) are 10-40 times those obtained with LAC1 and the redox potential of its T1 copper is 0.17 V lower than that of LAC1 (E degrees = 0.73 V). This is the first report on a single organism producing simultaneously both a high and a low redox potential laccase. The cDNA, clac2, was cloned and sequenced. It encodes a protein of 528 amino acids that shares 69% identity (79% similarity) with LAC1 and 81% identity (95% similarity) with Lcc3-2 from Polyporus ciliatus (AF176321-1), its nearest neighbor in database. Possible reasons for why this basidiomycete produces, in vivo, enzyme forms with such different behaviors are discussed.     

Characterization of a Ca2+-calmodulin-stimulated cyclic GMP phosphodiesterase from bovine brain

A calmodulin-stimulated form of cyclic nucleotide phosphodiesterase from bovine brain has been extensively purified (1000-fold). Its specific activity is approximately 4 mumol min-1 (mg of protein)-1 when 1 microM cGMP is used as the substrate. This form of calmodulin-sensitive phosphodiesterase activity differs from those purified previously by showing a very low maximum hydrolytic rate for cAMP vs. cGMP. The purification procedure utilizing ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephacryl S-300, isoelectric focusing, and affinity chromatography on calmodulin-Sepharose and Cibacron blue-agarose results in a protein with greater than 80% purity with 1% yield. Kinetics of cGMP and cAMP hydrolysis are linear with Km values of 5 and 15 microM, respectively. Addition of calcium and calmodulin reduces the apparent Km for cGMP to 2-3 microM and increases the Vmax by 10-fold. cAMP hydrolysis shows a similar increase in Vmax with an apparent doubling of Km. Both substrates show competitive inhibition with Ki's close to their relative Km values. Highly purified preparations of the enzyme contain a major protein band of Mr 74 000 that best correlates with enzyme activity. Proteins of Mr 59 000 and Mr 46 000 contaminate some preparations to varying degrees. An apparent molecular weight of 150 000 by gel filtration suggests that the enzyme exists as a dimer of Mr 74 000 subunits. Phosphorylation of the enzyme preparation by cAMP-dependent protein kinase did not alter the kinetic or calmodulin binding properties of the enzyme. Western immunoblot analysis indicated no cross-reactivity between the bovine brain calmodulin-stimulated gGMP phosphodiesterase and the Mr 60 000 high-affinity cAMP phosphodiesterase present in most mammalian tissues.     

Aldehyde oxidation in human placenta. Purification and properties of 1-pyrroline-5-carboxylate dehydrogenase.

The human placenta contains a considerable amount of 1-pyrroline-5-carboxylate dehydrogenase (23 +/- 6 micrograms/g; n = 12), about 25% of the concentration present in liver. The enzyme is the only form in placenta that oxidizes short- and medium-chain aldehydes, which facilitates its purification from this organ. It can be purified to homogeneity by successive chromatographies on DEAE-cellulose, 5'-AMP-Sepharose and Sephacryl S-300. From 500 g of tissue, about 2.1 units of enzyme can be obtained with a 12% yield. Placental 1-pyrroline-5-carboxylate dehydrogenase is a dimer of Mr-63,000 subunits. It exhibits a pI of 6.80-6.65, and is specific for 1-pyrroline-5-carboxylate, the cyclic form of glutamate gamma-semialdehyde (Km = 0.17 mM, kcat. = 870 min-1), although it also oxidizes short-chain aliphatic aldehydes such as propionaldehyde (Km = 24 mM, kcat. = 500 min-1). These properties are very close to those of the liver enzyme, indicating a strong similarity between the enzyme forms from both organs. The enzyme is highly sensitive to temperature, showing 50% inhibition after incubation for 0.8 min at 45 degrees C or after 23 min at 25 degrees C. It is irreversibly inhibited by disulfiram, and a molar ratio inhibitor: enzyme of 60:1 produced 50% inhibition after incubation for 10 min. A subcellular-distribution study indicates that the enzyme is located in two compartments: the mitochondria, with 60% of the total activity, and the cytosol, with 40% activity. The physiological role of the enzyme in placental amino acid metabolism is discussed.     

Oxidation of thiodiglycol (2,2'-thiobis-ethanol) by alcohol dehydrogenase: comparison of human isoenzymes

Sulfur mustard is a chemical warfare agent that causes blistering of the skin and damages the eyes and airway after environmental exposure. We have previously reported that thiodiglycol (TDG, 2,2'-bis-thiodiethanol), the hydrolysis product of sulfur mustard, is oxidized by alcohol dehydrogenase (ADH) purified from horse liver or present in mouse liver and human skin cytosol. Humans express four functional classes of ADH composed of several different isozymes, which vary in their tissue distribution, some occurring in skin. To help us evaluate the potential contribution of the various human isozymes toward toxicity in skin and in other tissues, we have compared the catalytic activity of purified human class I alphaalpha-, beta1beta1-, beta2beta2-, and gamma1gamma1-ADH, class II pi-ADH, class III chi-ADH, and class IV sigma-ADH with respect to TDG oxidation and their relative sensitivities to inhibition by pyrazole. Specific activities toward TDG were 123, 79, 347, 647, and 12 nmol/min/mg for the class I alphaalpha-, beta1,beta1-, beta2beta2-, and gamma1gamma1-ADH and class II pi-ADH, respectively. TDG was not a substrate for class III chi-ADH. The specific activity of class IV sigma-ADH was estimated at about 1630 nmol/min/mg. 1 mM pyrazole, a potent inhibitor of class I ADH, inhibited the class I alphaalpha, beta1beta1, beta2beta2, and gamma1gamma1 ADH and class IV sigma-ADH by 83, 100, 56, 90, and 73%, respectively. The class I alphaalpha- and beta1beta1-ADH oxidized TDG with kcat/Km value of 7-8 mM(-1) min(-1), beta2beta2-ADH with a value 19 mM(-1) min(-1) and class I gamma1gamma1-ADH with a value of 176 mM(-1) min(-1). The kcat/Km value for class IV sigma-ADH was estimated at 4 mM(-1) min(-1). The activities of class IV sigma-ADH and class I gamma1gamma1-ADH are of significant interest because of their prevalence in eyes, lungs, stomach, and skin, all target organs of sulfur mustard toxicity.     

A highly active and oxidation-resistant subtilisin-like enzyme produced by a combination of site-directed mutagenesis and chemical modification

The subtilisins are known to be susceptible to chemical oxidation due to the conversion of Met222 into the corresponding sulfoxide. A number of derivatives with resistance towards oxidation have previously been prepared by replacement of this group with the other 19 amino acid residues. Unfortunately, the activities of these enzymes were of the order of 1-10% of that obtained with the wild-type enzyme. In contrast, the oxidation-labile cysteine mutant exhibited much higher activity, suggesting that this is associated with the presence of a sulphur atom in the amino acid at position 222. It is shown here that it is possible to maintain a sulphur atom in the amino acid at position 222 without the enzyme becoming labile towards oxidation. A subtilisin from Bacillus lentus, subtilisin 309, in which Met222 was replaced with a cysteinyl residue by site-directed mutagenesis was modified with thioalkylating reagents. Treatment of such enzyme derivatives with H2O2 revealed that their stabilities towards oxidation had increased significantly compared to both wild-type and unmodified [Cys222]subtilisin. One of the chemically modified enzyme derivatives, [Me-S-Cys222]subtilisin, exhibited a kcat/Km value of 56% of that obtained with the wild-type enzyme when assayed against the substrate Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2 (Suc, succinyl) and it exhibited 89% activity when tested in an assay with dimethyl casein as a substrate. The corresponding values obtained for unmodified [Cys222]subtilisin were lower, i.e. 39% for the dimethyl casein activity and 46% for the kcat/Km for the hydrolysis of Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2. This demonstrates the feasibility of replacing the oxidation-labile methionyl residue group in a subtilisin enzyme with a group stable towards oxidation without substantially reducing the activity.     

Human alcohol dehydrogenases and serotonin metabolism

Human liver alcohol dehydrogenases (ADH) may participate in serotonin (5-hydroxytryptamine) metabolism. Class I and II isozymes catalyze the oxidation of 5-hydroxytryptophol (5-HTOL) with kcat/Km values ranging from 10 to 100 mM-1 min-1 compared to 4-66 mM-1 min-1 for that of ethanol at pH 7.40, 25 degrees C. The product, 5-hydroxyindoleacetaldehyde, was purified as its semicarbazone and identified by mass spectrometry. Ethanol competitively inhibits 5-HTOL oxidation by beta 1 gamma 2 ADH with a Ki of 440 microM, a value similar to the Km of ethanol, 210 microM. The inhibition constants for 1,10-phenanthroline and 4-methylpyrazole are 20 microM and 80 nM respectively, essentially identical to those obtained with ethanol as substrate, 22 microM and 70 nM, respectively. The competition between ethanol and 5-HTOL for ADH can explain observations of ethanol induced changes in serotonin metabolism in vivo.     

Reaction of Escherichia coli and yeast photolyases with homogeneous short-chain oligonucleotide substrates

Similar rates have been observed for dimer repair with Escherichia coli photolyase and the heterogeneous mixtures generated by UV irradiation of oligothymidylates [UV-oligo(dT)n, n greater than or equal to 4] or DNA. Comparable stability was observed for ES complexes formed with UV-oligo(dT)n, (n greater than or equal to 9) or dimer-containing DNA. In this paper, binding studies with E. coli photolyase and a series of homogeneous oligonucleotide substrates (TpT, TpTp, pTpT, TpTpT, TpTpT, TpTpTpT, TpTpTpT, TpTpTpT, TpTpTpT) show that about 80% of the binding energy observed with DNA as substrate (delta G approximately 10 kcal/mol) can be attributed to the interaction of the enzyme with a dimer-containing region that spans only four nucleotides in length. This major binding determinant (TpTpTpT) coincides with the major conformational impact region of the dimer and reflects contributions from the dimer itself (TpT, delta G = 4.6 kcal/mol), adjacent phosphates (5'p, 0.8 kcal/mol; 3'p, 1.1 kcal/mol), and adjacent thymine residues (5'T, 0.8 kcal/mol; 3'T, 1.3 kcal/mol). Similar turnover rates (average kcat = 6.7 min-1) are observed with short-chain oligonucleotide substrates and UV-oligo(dT)18, despite a 25,000-fold variation in binding constants (Kd). In contrast, the ratio Km/Kd decreases as binding affinity decreases and appears to plateau at a value near 1. Turnover with oligonucleotide substrates occurs at a rate similar to that estimated for the photochemical step (5.1 min-1), suggesting that this step is rate determining. Under these conditions, Km will approach Kd when the rate of ES complex dissociation exceeds kcat.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a basic carboxypeptidase from human seminal plasma

A carboxypeptidase which cleaves the C-terminal arginine or lysine from peptides was purified by a two-step procedure; gel filtration on Sephacryl S-300 and affinity chromatography on arginine-Sepharose. The activity increased 280% after the first step, indicating the removal of an inhibitor from the crude starting material. The activity in the crude seminal plasma eluted from the Sephacryl S-300 column with an apparent Mr 98,000 and after purification with an Mr 67,000, indicating that it binds to another protein in the crude seminal plasma. When analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single band at Mr 53,000 was seen which was converted to two smaller bands (Mr 32,000 and/or 26,000) after reduction. The seminal plasma carboxypeptidase has a neutral pH optimum, is inhibited by o-phenanthroline and by the inhibitor of carboxypeptidase B-type enzymes, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and can be activated by cobalt. The purified enzyme has a high specific activity (67.8 mumol/min/mg) with the ester substrate benzoyl (Bz)-Gly-argininic acid and readily cleaves Bz-Ala-Lys, Bz-Gly-Arg, and Bz-Gly-Lys. It also hydrolyzes biologically active peptides such as bradykinin (Km = 6 microM, kcat = 43 min-1), Arg6-Met5-enkephalin (Km = 103 microM, kcat = 438 min-1), and Lys6-Met5-enkephalin (Km = 848 microM, kcat = 449 min-1). The seminal plasma carboxypeptidase did not cross-react with antiserum to human plasma carboxypeptidase N; other properties distinguish it from the blood plasma enzyme as well as from pancreatic carboxypeptidase B and granular, acid carboxypeptidase H (enkephalin convertase). The carboxypeptidase could be involved in the control of fertility by activating or inactivating peptide hormones in the seminal plasma. In addition it could contribute to the degradation of basic proteins during semen liquefaction.     

Human carboxypeptidase M. Purification and characterization of a membrane-bound carboxypeptidase that cleaves peptide hormones

A membrane-bound neutral carboxypeptidase B-like enzyme was solubilized from human placental microvilli with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and purified to homogeneity by ion-exchange chromatography and affinity chromatography on arginine-Sepharose. It gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 62,000 with or without reduction. The enzyme is a glycoprotein as shown by its high affinity for concanavalin A-Sepharose and reduction in mass to 47,600 daltons after chemical deglycosylation. It has a neutral pH optimum, is activated by CoCl2, and inhibited by o-phenanthroline, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, or cadmium acetate, indicating it is a metallopeptidase. The enzyme cleaves arginine or lysine from the COOH terminus of synthetic peptides (e.g. Bz-Gly-Arg, Bz-Gly-Lys, Bz-Ala-Lys, dansyl-Ala-Arg, where Bz is benzoyl and dansyl is 5-dimethylaminonaphthalene-1-sulfonyl) as well as from several biologically active substrates: dynorphin A(1-13), Met5-Arg6-enkephalin (Km = 46 microM, kcat = 934 min-1), bradykinin (Km = 16 microM, kcat = 147 min-1), Met5-Lys6-enkephalin (Km = 375 microM, kcat = 663 min-1), and Leu5-Arg6-enkephalin (Km = 63 microM, kcat = 106 min-1). Although the enzyme shares some properties with other carboxypeptidase B-like enzymes, it is structurally, catalytically, and immunologically distinct from pancreatic carboxypeptidase A or B, human plasma carboxypeptidase N, and carboxypeptidase H ("enkephalin convertase"). To denote that the enzyme is membrane-bound, and to distinguish it from other known carboxypeptidases, we propose the name "carboxypeptidase M." Because of its localization on the plasma membrane and optimal activity at neutral pH, carboxypeptidase M could inactivate or modulate the activity of peptide hormones either before or after their interaction with plasma membrane receptors.