Publications

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

  Henseling J., Rohm K.H.

  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 studi ... >> More

  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. << Less

  Biochim Biophys Acta 959:370-377(1988) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

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

  Heese D., Berger S., Rohm K.H.

  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 re ... >> More

  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. << Less

  Eur J Biochem 188:175-180(1990) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Essential roles of zinc ligation and enzyme dimerization for catalysis in the aminoacylase-1/M20 family.

  Lindner H.A., Lunin V.V., Alary A., Hecker R., Cygler M., Menard R.

  Members of the aminoacylase-1 (Acy1)/M20 family of aminoacylases and exopeptidases exist as either monomers or homodimers. They contain a zinc-binding domain and a second domain mediating dimerization in the latter case. The roles that both domains play in catalysis have been investigated for huma ... >> More

  Members of the aminoacylase-1 (Acy1)/M20 family of aminoacylases and exopeptidases exist as either monomers or homodimers. They contain a zinc-binding domain and a second domain mediating dimerization in the latter case. The roles that both domains play in catalysis have been investigated for human Acy1 (hAcy1) by x-ray crystallography and by site-directed mutagenesis. Structure comparison of the dinuclear zinc center in a mutant of hAcy1 reported here with dizinc centers in related enzymes points to a difference in zinc ligation in the Acy1/M20 family. Mutational analysis supports catalytic roles of zinc ions, a vicinal glutamate, and a histidine from the dimerization domain. By complementing different active site mutants of hAcy1, we show that catalysis occurs at the dimer interface. Reinterpretation of the structure of a monomeric homolog, peptidase V, reveals that a domain insertion mimics dimerization. We conclude that monomeric and dimeric Acy1/M20 family members share a unique active site architecture involving both enzyme domains. The study may provide means to improve homologous carboxypeptidase G2 toward application in antibody-directed enzyme prodrug therapy. << Less

  J. Biol. Chem. 278:44496-44504(2003) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• The distribution of aminoacylase I among mammalian species and localization of the enzyme in porcine kidney.

  Lindner H., Hopfner S., Tafler-Naumann M., Miko M., Konrad L., Rohm K.H.

  Aminoacylase I (Acy-1, EC 3.5.1.14) is found in many mammalian tissues, with highest activities occurring in kidney. The enzyme hydrolyzes a variety of N-acylated amino acids; however, the physiological role and the exact cellular localization of Acy-1 are still a matter of debate. The comparison ... >> More

  Aminoacylase I (Acy-1, EC 3.5.1.14) is found in many mammalian tissues, with highest activities occurring in kidney. The enzyme hydrolyzes a variety of N-acylated amino acids; however, the physiological role and the exact cellular localization of Acy-1 are still a matter of debate. The comparison of Acy-1 activities in kidney and liver homogenates of 11 mammalian species showed that the enzyme is most abundant in true herbivores such as sheep and cattle as well as in omnivores, while activities were very low in both rodents and the cat. Acy-1 activity was not detected in livers of dogs of five different breeds. Using in situ hybridization of porcine kidney sections with DIG-labeled RNA probes, Acy-1 mRNA was shown to be evenly distributed throughout the tubular system, while glomeruli and the interstitium were free of stain. During subcellular fractionation, porcine Acy-1 behaved like a typical cytosolic enzyme. Commonly, Acy-1 is thought to catalyze hydrolytic reactions, i.e., the formation of free amino acids from acylated derivatives. Based on the present results and literature data, we propose a novel hypothesis, i.e., that Acy-1 catalyzes the synthesis (rather than the hydrolysis) of hippurate that is formed as a detoxification product of aromatic compounds. << Less

  Biochimie 82:129-137(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Site-directed mutagenesis and molecular modelling studies show the role of Asp82 and cysteines in rat acylase 1, a member of the M20 family.

  Herga S., Brutus A., Vitale R.M., Miche H., Perrier J., Puigserver A., Scaloni A., Giardina T.

  Acylase 1 from rat kidney catalyzes the hydrolysis of acyl-amino acids. Sequence alignment has shown that this enzyme belongs to the metalloprotein family M20. Site-directed mutagenesis experiments led to the identification of one functionally important amino acid residue located near one of the z ... >> More

  Acylase 1 from rat kidney catalyzes the hydrolysis of acyl-amino acids. Sequence alignment has shown that this enzyme belongs to the metalloprotein family M20. Site-directed mutagenesis experiments led to the identification of one functionally important amino acid residue located near one of the zinc coordinating residues, which play a critical role in the enzymatic activity. The D82N- and D82E-substituted forms showed no significant activity and very low activity, respectively, along with a loss of zinc coordination. Molecular modelling investigations indicated a putative role of D82 in ensuring a proper protonation of catalytic histidine. In addition, none of the five cysteine residues present in the rat kidney acylase 1 sequence seemed involved in the catalytic process: the loss of activity induced by the C294A substitution was probably due to a conformational change in the 3D structure. << Less

  Biochem Biophys Res Commun 330:540-546(2005) [PubMed] [EuropePMC]

• Roles of dimerization domain residues in binding and catalysis by aminoacylase-1.

  Lindner H.A., Alary A., Boju L.I., Sulea T., Menard R.

  The aminoacylase-1/metallopeptidase 20 (Acy1/M20) family is the largest metallopeptidase family. Several crystal structures feature a metal-binding and a dimerization-mediating domain, both arranged in an extended open conformation. We have recently shown [Lindner et al. (2003) J. Biol. Chem. 278, ... >> More

  The aminoacylase-1/metallopeptidase 20 (Acy1/M20) family is the largest metallopeptidase family. Several crystal structures feature a metal-binding and a dimerization-mediating domain, both arranged in an extended open conformation. We have recently shown [Lindner et al. (2003) J. Biol. Chem. 278, 44496-44504] that in human Acy1 the invariant residues Glu147 and His206 from the metal-binding and the dimerization domain, respectively, are recruited to the active site from opposite dimer subunits. We hypothesized that, to facilitate this, formation of the binary complex is associated with domain closure, which would also position additional residues in the functional active site of Acy1. These would include two partially conserved dimerization domain residues: an asparagine (Asn263) and an arginine (Arg276) from the same subunit as His206 and Glu147, respectively. In this paper, we investigate the significance of the three dimerization domain residues of human Acy1 His206, Asn263, and Arg276 and, additionally, the nearby Asp274 for catalysis using site-directed mutagenesis. Enzyme complementation assays confirm the putative subunit allocations of these residues, and steady-state kinetics support roles for all of them in catalysis but only involve the Arg276 in substrate-binding. The results are consistent with a model of the closed conformation for the structure of the related enzyme carboxypeptidase G2. This study demonstrates experimentally for the first time for a member of the Acy1/M20 family that several residues outside of the metal-binding domain are involved in binding and catalysis. << Less

  Biochemistry 44:15645-15651(2005) [PubMed] [EuropePMC]

• Aminoacylase I from porcine kidney: identification and characterization of two major protein domains.

  Palm G.J., Rohm K.H.

  The domain structure of hog-kidney aminoacylase I was studied by limited proteolytic digestion with trypsin and characterization of the resulting fragments. In the native enzyme, the sequences from residue 6 to 196 and 307 to 406 are resistant to trypsin and remain tightly bound in nondenaturing s ... >> More

  The domain structure of hog-kidney aminoacylase I was studied by limited proteolytic digestion with trypsin and characterization of the resulting fragments. In the native enzyme, the sequences from residue 6 to 196 and 307 to 406 are resistant to trypsin and remain tightly bound in nondenaturing solvents, while the intervening sequence (197-306) is efficiently degraded by trypsin. We conclude that the N-terminal half of the molecule and its C-terminal fourth form two independently folded domains. Both contain a peculiar PWW(A,L) sequence motif preceded by several strongly polar residues. We propose that these sequences form surface loops that mediate the membrane association of aminoacyclase I. We further show that the three free cysteine residues and the essential Zn2+ ion reside in the trypsin-resistant domains, while the intervening sequence contains the only disulfide H bond of the protein. << Less

  J Protein Chem 14:233-240(1995) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Acylase I-catalyzed deacetylation of N-acetyl-L-cysteine and S-alkyl-N-acetyl-L-cysteines.

  Uttamsingh V., Keller D.A., Anders M.W.

  The aminoacylase that catalyzes the hydrolysis of N-acetyl-L-cysteine (NAC) was identified as acylase I after purification by column chromatography and electrophoretic analysis. Rat kidney cytosol was fractionated by ammonium sulfate precipitation, and the proteins were separated by ion-exchange c ... >> More

  The aminoacylase that catalyzes the hydrolysis of N-acetyl-L-cysteine (NAC) was identified as acylase I after purification by column chromatography and electrophoretic analysis. Rat kidney cytosol was fractionated by ammonium sulfate precipitation, and the proteins were separated by ion-exchange column chromatography, gel-filtration column chromatography, and hydrophobic interaction column chromatography. Acylase activity with NAC and N-acetyl-L-methionine (NAM), a known substrate for acylase I, as substrates coeluted during all chromatographic steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the protein was purified to near homogeneity and had a subunit Mr of 43 000, which is identical with the Mr of acylase I from porcine kidney and bovine liver. n-Butylmalonic acid was a slow-binding inhibitor of acylase I and inhibited the deacetylation of NAC with a Ki of 192 +/-27 microM. These results show that acylase I catalyzes the deacetylation of NAC. The acylase I-catalyzed deacetylation of a range of S-alkyl-N-acetyl-L-cysteines, their carbon and oxygen analogues, and the selenium analogue of NAM was also studied with porcine kidney acylase I. The specific activity of the acylase I-catalyzed deacetylation of these substrates was related to their calculated molar volumes and log P values. The S-alkyl-N-acetyl-L-cysteines with short (C0-C3) and unbranched S-alkyl substituents were good acylase I substrates, whereas the S-alkyl-N-acetyl-L-cysteines with long (>C3) and branched S-alkyl substituents were poLr acylase I substrates. The carbon and oxygen analogues of S-methyl-N-acetyl-L-cysteine and the carbon analogue of S-ethyl-N-acetyl-L-cysteine were poor acylase I substrates, whereas the selenium analogue of NAM was a good acylase I substrate. << Less

  Chem Res Toxicol 11:800-809(1998) [PubMed] [EuropePMC]