A new family of carbon-nitrogen hydrolases.

Using computer methods for database search and multiple alignment, statistically significant sequence similarities were identified between several nitrilases with distinct substrate specificity, cyanide hydratases, aliphatic amidases, beta-alanine synthase, and a few other proteins with unknown molecular function. All these proteins appear to be involved in the reduction of organic nitrogen compounds and ammonia production. Sequence conservation over the entire length, as well as the similarity in the reactions catalyzed by the known enzymes in this family, points to a common catalytic mechanism. The new family of enzymes is characterized by several conserved motifs, one of which contains an invariant cysteine that is part of the catalytic site in nitrilases. Another highly conserved motif includes an invariant glutamic acid that might also be involved in catalysis.     

Quantitative distribution of lysosomal hydrolases in the rat nephron.

The activities of N-acetyl-beta, D-glucosaminidase (NAG, EC 3.2.1.30), beta, D-galactosidase (beta-gal, EC 3.2.1.23) and acid phosphatase (ac-Pase, EC 3.1.3.2) were measured in the glomeruli, five segments of the proximal and four segments of the distal tubule of normal male Wistar rats. The activities of NAG and beta-gal are 3- to 5-fold higher in the first part of the proximal tubule than in other segments and very low in glomeruli. We propose that the distribution of these two glycosidases reflects the contribution of the different tubular segments to the reabsorption of glycoproteins. The maximal activity of ac-Pase was found in the straight part of the proximal tubule. It was only 1.5-fold higher than in the distal tubule. Moreover, the activity in glomeruli is rather high. We conclude that ac-Pase is not primarily involved in the handling of reabsorbed molecules.     

Structural basis for the specificity of ubiquitin C-terminal hydrolases.

The release of ubiquitin from attachment to other proteins and adducts is critical for ubiquitin biosynthesis, proteasomal degradation and other cellular processes. De-ubiquitination is accomplished in part by members of the UCH (ubiquitin C-terminal hydrolase) family of enzymes. We have determined the 2.25 A resolution crystal structure of the yeast UCH, Yuh1, in a complex with the inhibitor ubiquitin aldehyde (Ubal). The structure mimics the tetrahedral intermediate in the reaction pathway and explains the very high enzyme specificity. Comparison with a related, unliganded UCH structure indicates that ubiquitin binding is coupled to rearrangements which block the active-site cleft in the absence of authentic substrate. Remarkably, a 21-residue loop that becomes ordered upon binding Ubal lies directly over the active site. Efficiently processed substrates apparently pass through this loop, and constraints on the loop conformation probably function to control UCH specificity.     

Molecular and immunological characterization of ADP-ribosylarginine hydrolases.

Mono-ADP-ribosylation is a reversible modification of proteins with NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyzing the forward and reverse reactions, respectively. Hydrolase activities were present in a variety of animal species, with the highest specific activities found in rat and mouse brain, spleen, and testis. Rat and mouse hydrolases were dithiothreitol- and Mg(2+)-dependent, whereas the bovine and guinea pig enzymes were dithiothreitol-independent. A rat brain hydrolase was purified approximately 20,000-fold and represented the major approximately 39-kDa protein on denaturing gels. Immunoaffinity-purified rabbit polyclonal antibodies reacted with 39-kDa proteins from turkey erythrocytes and rat, mouse, and calf brains. A rat brain cDNA library was screened using oligonucleotide and polymerase chain reaction-generated cDNA probes. Inserts from two overlapping clones yielded a composite sequence that included a 1086-base pair open reading frame, which contained amino acid sequences found in the purified hydrolase. A hydrolase fusion protein, synthesized in Escherichia coli, reacted with anti-39-kDa polyclonal antibodies and exhibited Mg(2+)- and dithiothreitol-dependent hydrolase activity. A coding region cDNA hybridized readily to a 1.7-kilobase band in rat and mouse poly(A)+ RNA, but poorly to bovine, chicken, rabbit, and human poly(A)+ RNA. The immunological and molecular biological data are consistent with partial conservation of hydrolase structure across animal species.     

Purification of glycoside hydrolases from Bacteroides fragilis.

Six glycoside hydrolases in the culture medium of Bacteroides fragilis--alpha-glucosidase, beta-glucosidase, alpha-galactosidase, beta-galactosidase, beta-N-acetylglucosaminidase, and alpha-L-fucosidase-were systematically purified by ammonium sulfate precipitation, gel filtration chromatography, and density gradient isoelectric focusing. The isoelectric focusing resolved the glycosidases into distinct, well-separated fractions and revealed three differently charged forms of beta-N-acetylglucosaminidase and of alpha-L-fucosidase. Furthermore, alpha-glucosidase and beta-N-acetylglucosaminidase were shown to possess dual affinities for the respective galactoside substrates, and beta-galactosidase also hydrolyzed beta-D-fucoside. alpha-Glucosidase was purified to homogeneity, as indicated by a thin-layer isoelectric focusing zymogram technique. The glycosidases, with exception of beta-glucosidase and the acid alpha-L-fucosidase, were each separated from other glycosidic activities to 99%. The molecular weights varied between 58,000 and 125,000. The pH optima ranged from 4.8 to 6.9.     

Structural equivalents of latency for lysosome hydrolases.

1. Structure-linked latency, a trait for most lysosome hydrolase activities, is customarily ascribed to the permeability-barrier function performed by the particle-limiting membrane, which shields enzyme sites from externally added substrates. 2. The influence of various substrate concentrations on the reaction rate has been measured for both free (non-latent) and total (completely unmasked by Triton X-100) hydrolase activities in rat liver cell-free preparations. The substrates were: beta-glycerophosphate, phenolphthalein mono-beta-glucuronide. p-nitrophenyl N-acetyl-beta-D-glucosaminide and p-nitrophenyl beta-D-galactopyranoside. The ratio (free activity/total activity) X 100 is called fractional free activity at any given substrate concentration. 3. The fractional free activity of beta-glucuronidase and beta-N-acetylglucosaminidase were clearly independent of substrate concentration, over the range examined, in both homogenates and lysosome-rich fractions. The fractional free activity of acid phosphatase appeared to be either unaffected (homogenate) or even depressed (lysosome-rich fraction) by increasing the beta-glycerophosphate concentration. The fractional free activity of beta-galactosidase consistently showed a non-linear increase with increasing substrate concentration in both homogenates and lysosome-rich fractions. 4. Procedures such as treatment with digitonin, hypo-osmotic shock and acid autolysis, although effective in causing varying degrees of resolution of the latency of lysosome hydrolase activities, were unable to modify appreciably the pattern of dependence or independence of their fractional free activities on substrate concentration, as compared with that exhibited by control preparations. Ouabain did not affect the free beta-N-acetylglucosaminidase activity of liver homogenates at all. 5. Preincubation of control preparations with beta-glycerophosphate or p-nitrophenyl beta-galactoside did not result in any significant stimulation of the free hydrolytic activity toward these substrates. 6. The results consistently support the view that the membrane of "intact" lysosomes is virtually impermeable to all the substrates tested, except for p-nitrophenyl beta-galactoside, for which the evidence is contradictory. Moreover the progressive unmasking of the hydrolase activities produced by these procedures in vitro reflects the increasing proportion of enzyme sites that are fully accessible to their substrates rather than a graded increase in the permeability of the lysosomal membrane.     

Nitrile hydrolases

A number of nitrile-related enzymes have been screened over the past year for use in synthetic applications. There have also been significant advances in our understanding of the structures and modes of regulation of metal-containing nitrile hydratases. Enzyme structural characterization has provided new insights into how the molecular structure determines the enzyme function and how an enzyme can be endowed with new properties. This information has important implications for potential future applications other than the present industrial production of acrylamide and nicotinamide.     

Isolation and characterisation of peptide hydrolases from the maize root.

The maize root has two main proteinase and carboxypeptidase components. Proteinase I and carboxypeptidase I, which predominate in older plants, appear to have a serine group at their active sites and have been estimated to have molecular weights of approximately 54000 and 77000 respectively. Proteinase I, which has been purified up to 500-fold, degrades haemoglobin and azocasein with maximum activity at pH 4 and 9--10 respectively, while on maize root protein it gives most hydrolysis in the neutral pH range. The main portion of the nitrate-reductase-inactivating activity in the maize root extract is due to proteinase I. Carboxypeptidase I, like several other plant carboxypeptidases such as carboxypeptidase C which have now (IUB Recommendations 1978) been classified as serine carboxypeptidases (EC 3.4.16.1), has maximum activity around pH 5 and has esterase activity. A second group of proteases, proteinase II and carboxypeptidase II, separated from the above on carboxymethyl-cellulose, were shown to have different molecular weight properties and be equally sensitive to serine and thiol group inhibitors. Proteinase II degrades haemoglobin, but not azocasein and does not mediate nitrate reductase inactivation. Associated with this second group of proteases was a macromolecular component which inactivated nitrate reductase but, unlike the action of proteinase I, was not inhibited by phenylmethylsulphonyl fluoride or casein. It was inhibited by metal chelating agents which were without effect on nitrate reductase inactivation due to proteinase I.     

ADP-ribosylarginine hydrolases

ADP-ribosylation is a reversible post-translational modification of proteins involving the addition of the ADP-ribose moiety of NAD to an acceptor protein or amino acid. NAD: arginine ADP-ribosyltransferase, purified from numerous animal tissues, catalyzes the transfer of ADP-ribose to an arginine residue in proteins. The reverse reaction, catalyzed by ADP-ribosylarginine hydrolase, removes ADP-ribose, regenerating free arginine. An ADP-ribosylarginine hydrolase, purified extensively from turkey erythrocytes, was a 39-kDa monomeric protein under denaturing and non-denaturing conditions, and was activated by Mg²⁺ and dithiothreitol. The ADP-ribose moiety was critical for substrate recognition; the enzyme hydrolyzed ADP-ribosylarginine and (2-phospho-ADP-ribosyl)arginine but not phosphoribosylarginine or ribosylarginine. The hydrolase cDNA was cloned from rat and subsequently from mouse and human brain. The rat hydrolase gene contained a 1086-base pair open reading frame, with deduced amino acid sequences identical to those obtained by amino terminal sequencing of the protein or of HPLC-purified tryptic peptides. Deduced amino acid sequences from the mouse and human hydrolase cDNAs were 94% and 83% identical, respectively to the rat. Anti-rat brain hydrolase polyclonal antibodies reacted with turkey erythrocyte, mouse and bovine brain hydrolase. The rat hydrolase, expressed inE. coli, demonstrated enhanced activity in the presence of Mg²⁺ and thiol, whereas the recombinant human hydrolase was stimulated by Mg²⁺ but was thiol-independent. In the rat and mouse enzymes, there are five cysteines in identical positions; four of the cysteines are conserved in the human hydrolase. Replacement of cysteine 108 in the rat hydrolase (not present in the human enzyme) resulted in a thiol-independent hydrolase without altering specific activity. Rabbit anti-rat brain hydrolase antibodies reacted on immunoblot with the wild-type rat hydrolase and only weakly with the mutant hydrolase. There was no immunoreactivity with either the wild-type or mutant human enzyme. Cysteine 108 in the rat and mouse hydrolase may be responsible in part for thiol-dependence as wall as antibody recognition. Based on these studies, the mammalian and avian ADP-ribosylarginine hydrolases exhibit considerable conservation in structure and function.     

Identification of yolk platelet-associated hydrolases in the oocytes of Rhodnius prolixus.

The yolk platelets from Rhodnius prolixus, a blood-sucking bug, are composed mostly of vitellin and here are shown to contain at least two hydrolytic enzymes, a phosphatase and a cathepsin D-like proteinase. Both the proteinase and the phosphatase have an acid pH optimum. No hydrolytic activity was observed under alkaline or neutral conditions. Among several proteinase inhibitors tested, only pepstatin could abolish vitellin breakdown in vitro. The proteinase appears to be bound to the yolk platelet membranes. The phosphatase activity, using p-nitrophenyl phosphate as substrate, was enhanced after disruption of the platelet membrane by Triton X-100. This activity could be inhibited by tartrate but not by p-cloromercuribenzoate.     

Isolation of palmitoyl-CoA hydrolases from human blood platelets.

The palmitoyl-CoA hydrolase activity, which in human blood platelets is mainly localized in the cytosol fraction [Berge, Vollset & Farstad (1980) Scand. J. Clin. Lab. Invest. 40, 271--279], was found to be extremely labile. Inclusion of glycerol or palmitoyl-CoA stabilized the activity during preparation. Gel-filtration studies revealed multiple forms of the enzyme with molecular weights corresponding to about 70 000, 40 000 and 24 000. The relative recovery of the mol.wt.-70 000 form was increased by the presence of 20% (v/v) glycerol or 10 microM-palmitoyl-CoA. The three enzyme forms are probably unrelated, since they were not interconvertible. The three different species of palmitoyl-CoA hydrolase were purified by DEAE-cellulose and hydroxyapatite chromatography, isoelectric focusing and high-pressure liquid chromatography (h.p.l.c.) to apparent homogeneity. The three enzymes had isoelectric points (pI) of 7.0, 6.1 and 4.9. The corresponding molecular weights were 27 000--33 000, 66 000--72 000 and 45 000--49 000, calculated from h.p.l.c. and Ultrogel AcA-44 chromatography. The apparently purified enzymes were unstable, as most of the activity was lost during purification. The enzyme with an apparent molecular weight of 45 000--49 000 was split into fractions with molecular weights of less than 10 000 by re-chromatography on h.p.l.c. concomitantly with a loss of activity. The stimulation of the activity by the presence of serum albumin seems to depend on the availability of palmitoyl-CoA, as has been reported for other palmitoyl-CoA hydrolases. [Berge & Farstad (1979) Eur. J. Biochem. 96, 393--401].     

Binding of human liver hydrolases by immobilized lectins.

The binding of 22 human liver hydrolase activities by immobilized lectins of six different carbohydrate specificities, namely alpha-D-mannose (glucose), D-N-acetylglucosamine, D-N-acetylgalactosamine, L-fucose, alpha-D-galactose and beta-D-galactose, were examined. Differences in binding among these enzymes and within specific enzymes were observed. For example, the neutral forms of alpha-mannosidase and beta-xylosidase were bound by the Ulex europaeus lectin I (specific for L-fucose), whereas the acidic forms were not. Bandierea simplicifolia lectin (specific for alpha-galactose) bound 65% of beta-glucuronidase activity; recycling experiments demonstrated complete binding of the enzyme that had been eluted with the competitor D-galactose and no binding of the fraction that was not initially bound. These results suggested the presence of two forms of this enzyme. Similar data were obtained for acidic beta-galactosidase activity. These experiments may provide the basis for the expanded use of immobilized lectins for purification and characterization of hydrolases and other glycoproteins.