Aminoacylase I deficiency: a novel inborn error of metabolism

This is the first report of a patient with aminoacylase I deficiency. High amounts of N-acetylated amino acids were detected by gas chromatography-mass spectrometry in the urine, including the derivatives of serine, glutamic acid, alanine, methionine, glycine, and smaller amounts of threonine, leucine, valine, and isoleucine. NMR spectroscopy confirmed these findings and, in addition, showed the presence of N-acetylglutamine and N-acetylasparagine. In EBV transformed lymphoblasts, aminoacylase I activity was deficient. Loss of activity was due to decreased amounts of aminoacylase I protein. The amount of mRNA for the aminoacylase I was decreased. DNA sequencing of the encoding ACY1 gene showed a homozygous c.1057 C>T transition, predicting a p.Arg353Cys substitution. Both parents were heterozygous for the mutation. The mutation was also detected in 5/161 controls. To exclude the possibility of a genetic polymorphism, protein expression studies were performed showing that the mutant protein had lost catalytic activity.     

Nuclear magnetic relaxation studies of the role of the metal ion in Mn2(+)-substituted aminoacylase I

Substitution of the essential Zn2+ ions of porcine kidney aminoacylase I (EC 3.5.1.14) by Mn2+ did not markedly affect the kinetic properties of the enzyme. Using Mn2+ as a paramagnetic probe, we were able to study the conformations of bound ligands by measuring the enhancement of ligand proton relaxation in 1H NMR. In addition, the effects of inhibitors on the paramagnetic enhancement of water proton relaxation rates were examined. The results of both approaches, in agreement with kinetic evidence, suggest that the metal center of aminoacylase I is too distant from the ligand binding site to allow direct participation of the metal in substrate binding or catalysis. We, therefore, propose that the metal ion of aminoacylase I plays a purely structural role.     

Aminoacylase pellets.

Aminoacylase was immobilized on the mycelium pellets of Aspergillus ochraceus by using albumin and glutaraldehyde. No difference in the optimum pH was observed between native aminoacylase and aminoacylase pellets. The aminoacylase pellets were stable in pH 4-8 but they were unstable in alkaline conditions. The aminoacylase pellets were more stable against heavy metal ions and inhibitors than native aminoacylase. However, the degree of the activation of aminoacylase with cobalt ion decreased with the immobilization. It was suggested that most of aminoacylase was covalently coupled to the mycelium with glutaraldehyde.     

Aminoacylase I from hog kidney: anion effects and the pH dependence of kinetic parameters

The hydrolysis of acetylamino acids by highly purified hog kidney aminoacylase I (N-acylamino acid amidohydrolase, EC 3.5.1.14) was investigated using flow injection analysis to determine reaction rates. We show that the distinctly bell-shaped pH versus activity profiles observed in previous studies do not reflect protonic equilibria in the enzyme, but were created by buffer effects. At low pH, anions such as phosphate, nitrate or chloride markedly increase Km. These effects are reversed at higher pH. In zwitterionic 'Good' buffers (Mes, Mops, and Bicine), maximal velocities are almost independent of pH between 6.5 and 9 for all substrates studied (Ac-LAla, Ac-LGlu, Ac-LMet, Ac-LPhe). Below pH 6.5, the catalytic constants decrease with pH, apparently due to the protonation of a carboxylate with a pKa of 5.5-6. The pH dependence of Km markedly varies among different substates. We conclude that the observed profiles all result from the dissociation of an active-site residue with a pKa of 8-8.5, which we tentatively identify as an active-site cysteine residue. A working model of aminoacylase catalysis is presented that accounts for most of the known facts.     

Further characterization of porcine kidney aminoacylase I reveals close similarity to 'renal dipeptidase'

We present data indicating that aminoacylase I (EC 3.5.1.14) from porcine kidney and 'renal dipeptidase' (EC 3.4.13.11) are closely related. We show that, in situ, a considerable fraction of aminoacylase activity ist attached to membranes. Incubation of washed microsomal membranes with phospholipase C from B. cereus results in the rapid solubilization of aminoacylase I, suggesting that aminoacylase--as shown for renal dipeptidase before--bears a glycolipid 'membrane anchor'. In agreement with this assumption, purified aminoacylase was found to contain myo-inositol, a characteristic component of phosphatidylinositol-anchored membrane proteins. A reexamination of the molecular mass of purified aminoacylase yielded values (46,000 +/- 2,000 Da by SDS polyacrylamide electrophoresis, 98,000 +/- 5,000 Da by sedimentation equilibrium centrifugation) similar to those reported for renal dipeptidase. The enzymes coelute during most of the procedures applied in the purification of aminoacylase or renal dipeptidase, but can be separated by hydrophobic interaction chromatography. A survey of the literature revealed a series of additional features of aminoacylase I and renal dipeptidase (amino-acid composition, isoelectric points, metal dependence, and more) that are strikingly similar.     

Facile oxygen exchanges of phosphoenolpyruvate and preparation of [18O]phosphoenolpyruvate

Phosphoenolpyruvate when heated in acidic solution exchanges its phosphoryl and carboxyl oxygens rapidly and its enolic oxygen much more slowly with oxygens from water. The incorporation of 18O into phosphoenolpyruvate was measured by gas chromatography-mass spectrometry and phosphorus-31 nuclear magnetic resonance after heating in H218O at 98 degrees C. The rates of exchange of all six oxygens of phosphoenolpyruvate with water increase with increasing acidity, and the phosphoryl oxygens exchange more rapidly than the carboxyl oxygens. The rate of exchange of each oxygen of the phosphoryl group is 16-fold greater than the hydrolysis rate at 1 N HCl. This provides a simple and useful method for the synthesis of [18O]phosphoenolpyruvate highly enriched in its phosphoryl-group oxygens. An enrichment of 89% was obtained with a 50% yield. The [18O]-phosphoenolpyruvate showed a binomial distribution of 18O in the phosphoryl-group oxygens. The exchange may be explained by the reversible formation of a transient cyclic phosphate and, for exchange of the enolic oxygen, a transient acyl phosphate. Preparation of [18O]phosphoenolypyruvate from [18O]Pi by a chemical synthesis from beta-chlorolactate was not satisfactory because of drastic loss of 18O during the procedures used. Some loss of 18O also occurred during an enzymic synthesis with KCNO, [18O]Pi, carbamate kinase, and pyruvate kinase.     

Isolation and characterization of N-long chain acyl aminoacylase from Pseudomonas diminuta

N-Long chain acyl aminoacylase II (Enzyme II) catalyzing the hydrolysis of N-long chain acyl amino acids was purified about 2,000-fold from the cell extracts of Pseudomonas diminuta with 1.8% of activity yield. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis and the molecular weight was 220,000. Enzyme II differed from N-long chain acyl aminoacylase I (Enzyme I) in molecular weight, in substrate specificity, and in behavior toward temperature and pH. Enzyme II showed broader substrate specificity than Enzyme I and catalyzed the hydrolysis of lipoamino acids containing various amino acid residues, although Enzyme I was almost specific to the lipoamino acids containing L-glutamate. The extent of hydrolysis by Enzyme II reaction varied depending on the kinds of lipoamino acids and were: 100% for palmitoyl-L-glutamate, 91% for myristoyl-L-glutamate, 85% for lauroyl-L-glutamate, 54% for lauroyl-L-aspartate, 28% for stearoyl-L-glutamate and 17.5% for lauroyl-glycine.     

Regulation of fatty acid 18O exchange catalyzed by pancreatic carboxylester lipase. 1. Mechanism and kinetic properties.

The exchange of 18O between H2O and long-chain free fatty acids is catalyzed by pancreatic carboxylester lipase (EC 1.1.1.13). For palmitic, oleic, and arachidonic acid in aqueous suspension and for 13,16-cis,cis-docosadienoic acid (DA) in monomolecular films, carboxyl oxygens were completely exchanged with water oxygens of the bulk aqueous phase. With enzyme at either substrate or catalytic concentrations in the argon-buffer interface, the exchange of DA oxygens obeyed a random sequential mechanism, i.e., 18O,18O-DA in equilibrium with 18O,16O-DA in equilibrium with 16O,16O-DA. This indicates that the dissociation of the enzyme-DA complex is much faster than the rate-limiting step in the overall exchange reaction. Kinetic analysis of 18O exchange showed a first-order dependence on surface enzyme and DA concentrations, i.e., the reaction was limited by the acylation rate. The values of kcat/Km, 0.118 cm2 pmol-1 s-1, for the exchange reaction was comparable to that for methyl oleate hydrolysis and 5-fold higher than that for cholesteryl oleate hydrolysis in monolayers [Bhat, S., & Brockman, H. L. (1982) Biochemistry 21, 1547]. Thus, fatty acids are good "substrates" for carboxylester lipase. With substrate levels of carboxylester lipase in the interfacial phase, the acylation rate constant kcat/Km was 200-fold lower than that obtained with catalytic levels of enzyme. This suggests a possible restriction of substrate diffusion in the protein-covered substrate monolayer.     

The 18O isotope effect in 13C nuclear magnetic resonance spectroscopy: mechanistic studies on asparaginase from Escherichia coli

The mechanism of the enzyme asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) from Escherichia coli was examined using 13C NMR spectroscopy. The pH-dependent oxygen exchange reactions between water and aspartic acid were followed by use of the 18O isotope-induced shift of the resonance positions of directly bonded 13C nuclei. Both L-1- and L-1,4-[13C]aspartic acid were used in experiments with previously 18O-labeled aspartic acid, or in experiments involving the use of 18O-labeled solvent water. Asparaginase catalyzes a relatively efficient exchange between the oxygens of water and those on one carboxyl group of aspartic acid. Exchange at C-4 occurs rapidly but, within experimental error, no exchange at C-1 could be detected. These and related experiments involving the position of 18O incorporation during hydrolysis of aspartic acid beta-methyl ester are all consistent with possible acyl-enzyme mechanisms involving C-4, but do not support a free aspartic acid anhydride mechanism.     

A spectrophotometric rate assay of aminoacylase

Acetamidoacrylate, a synthetic N-acetyl unsaturated amino acid, was hydrolyzed to acetate, ammonia, and pyruvate by hog kidney, fungal, and bacterial aminoacylases. A spectrophotometric procedure for rate assay of aminoacylase has been established with this substrate on the basis of the simultaneous reduction of pyruvate with NADH and alanine dehydrogenase. This assay is linear with time and enzyme concentration and is useful for kinetic studies of aminoacylase. This procedure is not influenced significantly by amino and thiol compounds and metal ions, which interfere with the ninhydrin methods traditionally used. Alanine dehydrogenase can be replaced by lactate dehydrogenase in the reaction system.     

Butylmalonate is a transition state analogue for aminocylase I

Butylmalonate (butyl propanedioic acid) is a slow-binding inhibitor of porcine renal aminoacylase I (EC 3.5.1.14), causing transients of activity with half-times of more than 10 min. At 25°C and pH 7.0, the dissociation rate of the complex is approximately 6 × 10⁻⁴ s⁻¹, while the rate constant of complex formation is in the order of 20 M⁻¹·s⁻¹. In good agreement with these data, steady-state kinetics yield an estimated inhibition constant around 100 μM. Molecular mechanics calculations showed that conformation and charge distribution of butylmalonate are strikingly similar to those of the putative transition state of aminoacylase catalysis.     

Crude aminoacylase from aspergillus sp. is a mixture of hydrolases

A range of cross-linked enzyme aggregates (CLEAs) was prepared from commercially available aminoacylase I. Results from three test reactions showed that aminoacylase does not possess aminolysis or alcoholysis activity, both previously ascribed to this enzyme. This result was confirmed using aminoacylase purified by chromatographic techniques, which leads us to conclude that the previously observed acylations of esters and amines is due to other enzymes present as impurities in the crude aminoacylase I.     

A simplified purification method of aminoacylase I

Taking advantage that the stability of aminoacylase I increases in the presence of Co2+, a simplified method of its purification, which includes a heating step, is described. This method is easy, fast, and gives a good yield of the enzyme. The properties of aminoacylase I thus prepared are similar to those described in the literature.     

Characterization of phosphate oxygen exchange reactions catalyzed by myosin through measurement of the distribution of 18-O-labeled species

The change in the distribution of the phosphate species containing 0 to 4 18O oxygens per Pi was investigated during medium Pi equilibrium HOH exchange catalyzed by myosin subfragment 1. At 25 degrees C, a Pi molecule once bound loses an average of 3.9 of its original 4 oxygens prior to release which means that at least 100 reversals of the exchange reaction must have occurred. At 0 degrees C, only 3.4 of the 4 oxygens are lost prior to release indicating an average of 17 reversals. Distribution patterns are consistent with equivalent participation in the exchange reactions of all 4 oxygens of bound Pi. The intermediate exchange of Pi oxygens during hydrolysis of 18O-labeled ATP by myosin has also been investigated. The distribution of the product Pi species shows that there is an ATPase component in myosin preparations which hydrolyzes ATP without intermediate exchange. Presence of this component, which is likely a contaminating ATPase, provides a simple explanation of the apparent nonequivalence of phosphate oxygens which has been observed. When correction is made for this contaminant, characteristics of the myosin intermediate Pi equilibrium HOH exchange are similar to those of myosin subfragment 1 medium exchange, and intermediate exchange data are in much closer agreement with other kinetic measurements.     

Dynamic reversal of enzyme carboxyl group phosphorylation as the basis of the oxygen exchange catalyzed by sarcoplasmic reticulum adenosine triphosphatase.

Millisecond mixing and quenching experiments demonstrate an apparent t1/2 for the labeling of phosphorylated sarcoplasmic reticulum ATPase by 32Pi at pH 6 and 30 degrees C of 30 to 40 ms. Under the same conditions, the rate of exchange of water oxygens with inorganic phosphate (Pi) is about 40 mol of H2O exchanged with Pi per 10(6) g of protein per s. Theoretical equations are developed for the expected 32P-labeling pattern given various comparative rates of flux between Pi and the Michaelis complex and between the Michaelis complex and phosphorylated enzyme. The results show that the rapid reversal of the formation of the phosphorylated enzyme is a major source of the oxygen exchange and are consistent with such reversal being the only source.     

The guanidine like effects of arginine on aminoacylase and salt-induced molten globule state

Aminoacylase is a dimeric enzyme containing one Zn(2+) ion per subunit. The arginine (Arg)-induced unfolding of Holo-aminoacylase and Apo-aminoacylase has been studied by measurement of enzyme activity, fluorescence emission spectra and 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra. Besides being the most alkaline amino acid, the arginine molecule contains a positively charged guanidine group, similar to guanidine hydrochloride, and has been used in many refolding systems to suppress protein aggregation. Our results showed that arginine caused the inactivation and unfolding of aminoacylase, with no aggregation during denaturation. A comparison between the unfolding of aminoacylase in aqueous and HCl (pH 7.5) arginine solutions indicated that the guanidine group of arginine had protein-denaturing effects similar to those of guanidine hydrochloride, which might help us understand the mechanism by which arginine suppresses incorrect refolding. The results showed that arginine-denatured aminoacylase could be reactivated and refolded correctly, indicating that arginine is as good a denaturant as the guanidine or urea for study of protein unfolding and refolding. Both the intrinsic fluorescence and the ANS fluorescence spectra showed that the arginine-unfolded aminoacylase formed a molten globule state in the presence of KCl, suggesting that intermediates exist during aminoacylase refolding. The results for the Apo-aminoacylase followed were similar to those for the Holo-enzyme, suggesting that Holo- and Apo-aminoacylase might have a similar unfolding and refolding pathway.     

Properties of acylase preparations from an actinomycete culture

A comparative study of some physico-chemical properties of high-purified preparations of extracellular penicillin-V-acylase and aminoacylase, isolated from the actinomycete Streptoverticillium No 62, revealed the difference in pH and temperature optima, in the sensitivity to the ionic composition of buffer solutions, in the enzyme stability during storage. As for the aminoacylase preparation, its thermostability was studied at different pH values, as well as the effect of specific compounds was tested. Similar to other fungal enzymes, the aminoacylase possesses a wide substrate specificity, and by its stereospecificity can be related to L-aminoacylases, while penicillin-V-acylase is a high-specific enzyme, active against phenoxymethylpenicillin.     

Aminoacylase I is not a glycolipid-anchored ectoenzyme in pig kidney.

Subcellular fractionation of pig kidney cortex revealed that aminoacylase I (EC 3.5.1.14, N-acyl-L-amino-acid aminohydrolase) is predominantly a soluble enzyme with only 0.5% of the total activity being recovered in the membrane fraction. The aminoacylase I activity associated with the membrane preparations displayed neither rapid release following incubation with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis nor the distinctive differential pattern of detergent solubilization which was seen with glycosyl-phosphatidylinositol-anchored proteins (renal dipeptidase, alkaline phosphatase). When fractionated by phase separation in Triton X-114, integral membrane proteins of kidney microvillar membranes partitioned predominantly (greater than 90%) into the detergent-rich phase. In contrast, only 3.7% of aminoacylase I activity associated with microvillar membranes partitioned into the detergent-rich phase. Aminoacylase I activity of pig kidney would therefore appear to be a hydrophilic protein in nature and is not, as suggested previously, a G-PI-anchored integral membrane protein.     

Studies on Continuous Enzyme Reactions

The kinetics of hydrolysis of acetyl-dl-methionine in DEAE-cellulose-aminoacylase (EC 3.5.1.14) column and DEAE-Sephadex-aminoacylase column was studied.

The rate of hydrolysis of substrate was shown to be dependent on the flow rate and independent to the dimension of the enzyme column. The rate of hydrolysis of the substrate was equal in cases of down-ward flow and of up-ward flow. The deteriorated aminoacylase columns by long period operation were reactivated by the recharge of aminoacylase to them. The continuous enzyme reaction using an aminoacylase column was superior to the batch enzyme reaction using native aminoacylase.

The enzymatic properties of the water-insoluble aminoacylase prepared by linking mold aminoacylase (EC 3.5.1.14) to DEAE-Sephadex were studied and compared with those of the native aminoacylase.

Optimum pH values for hydrolysis of several substrates by the DEAE-Sephadex-amino-acylase complex (DSA-complex) shifted about 0.5~1.5 pH units more to the acid side than those by the native enzyme. On the effects of metal ions and inhibitors, substrate specificity, optical specificity and kinetic constants, no marked difference was observed between the native enzyme and the DSA-complex. Heat stability, optimum temperature and resistance towards proteases were increased by conversion from the native form to the insoluble enzyme. It was also observed that the DSA-complex was activated by urea.     

Effects of temperature and pH on the water exchange through erythrocyte membranes: Nuclear magnetic resonance studies

Summary The temperature and pH dependence of water exchange has been studied on isolated erythrocytes suspended in isotonic buffered solutions. At pH 7.4 a break in the Arrhenius plot of water exchange time at around 26°C was found. The mean value of the apparent activation energy of the water exchange time at temperatures higher than that of the discontinuity was 5.7 kcal/mole (±0.4); at lower temperatures the values of the apparent activation energy were below 1.4 kcal/mole. The pH dependence of water exchange time of isolated erythrocytes revealed a marked increase of the water exchange time values in the acid range of pH; a much smaller variation of the same parameter occurs between pH 7.0 and 8.0. These finding could be correlated with other processes involving erythrocyte membranes that showed similar pH and temperature dependence and were considered to indicate state transitions in the membranes. It is suggested that the temperature and pH effects on water diffusion indicate that conformational changes and cooperative effects are implicated in the mechanism of this transport process.Institute for Isotopic and Molecular Technology.