Biomedical subjects

K H Röhm

Publications and source records attributed to K H Röhm.

At least 19 recordsLinked to original sources

PubMed1996

Crystal structure and amino acid sequence of Wolinella succinogenes L-asparaginase.

The amino acid sequence and tertiary structure of Wolinella succinogenes L-asparaginase were determined, and were compared with the structures of other type-II bacterial L-asparaginases. Each chain of this homotetrameric enzyme consists of 330 residues. The amino acid sequence is 40-50% identical to the sequences of related proteins from other bacterial sources, and all residues previously shown to be crucial for the catalytic action of these enzymes are identical. Differences between the amino acid sequence of W. succinogenes L-asparaginase and that of related enzymes are discussed in terms of the possible influence on the substrate specificity. The overall fold of the protein subunit is almost identical to that observed for other L-asparaginases. Two fragments in each subunit, a very highly flexible loop (approximately 20 amino acids) that forms part of the active site, and the N-terminus (two amino acids), are not defined in the structure. The orientation of Thr14, a residue probably involved in the catalytic activity, indicates the absence of ligand in the active-site pocket. The rigid part of the active site, which includes the asparaginase triad Thr93-Lys 166-Asp94, is structurally very highly conserved with equivalent regions found in other type-II bacterial L-asparaginases.

Amino Acid Sequence↗

PubMed1996

A covalently bound catalytic intermediate in Escherichia coli asparaginase: crystal structure of a Thr-89-Val mutant.

Escherichia coli asparaginase II catalyzes the hydrolysis of L-asparagine to L-aspartate via a threonine-bound acyl-enzyme intermediate. A nearly inactive mutant in which one of the active site threonines, Thr-89, was replaced by valine was constructed, expressed, and crystallized. Its structure, solved at 2.2 A resolution, shows high overall similarity to the wild-type enzyme, but an aspartyl moiety is covalently bound to Thr-12, resembling a reaction intermediate. Kinetic analysis confirms the deacylation deficiency, which is also explained on a structural basis. The previously identified oxyanion hole is described in more detail.

Asparaginase↗

PubMed1995

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

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.

Amidohydrolases↗

PubMed1995

Thermostable aminoacylase from Bacillus stearothermophilus: significance of the metal center for catalysis and protein stability.

A thermostable aminoacylase (N-acylamino acid amidohydrolase, EC 3.5.1.14) from Bacillus stearothermophilus was overexpressed in E. coli and characterized with respect to metal content, metal dependence, heat stability, and quaternary structure. Like other enzymes of the aminoacylase family, native aminoacylase contains one Zn2+ ion per subunit. Several other transition metal ions (Co2+, Mn2+ and Cd2+) also sustain aminoacylase activity toward N-acetyl L-alanine with Cd2+ giving the highest turnover number. The stability constants of the respective metal complexes were estimated by activity measurements in metal buffer systems. Co2+ also acts as an activator mainly by lowering the Km for the substrate. These data and CD spectra obtained with the native and the metal-free enzyme suggest a predominantly structural role for the intrinsic metal ion of thermostable aminoacylase. In contrast to previous reports the enzyme behaved as a dimer in analytical gel filtration.

Amidohydrolases↗

PubMed1994

States and functions of tyrosine residues in Escherichia coli asparaginase II.

The importance of five tyrosine residues of Escherichia coli asparaginase II (EcA2) for catalysis and protein stability was examined by site-directed mutagenesis, chemical modification of wild-type and variant enzymes, and by thermodynamic studies of protein denaturation. While the tyrosine residue Y25 is directly involved in catalysis, the hydroxyl groups of residues Y181, Y250, Y289 and Y326 are not necessary for EcA2 activity. However, residues Y181 and Y326 are crucial for stabilization of the native EcA2 tetramer. pH titration curves showed that the active-site residue Y25 has a normal pKa while the C-terminal Y326 is unusually acidic. 1H-NMR signals of a peculiar ligand-sensitive tyrosine residue were assigned to Y25. These and other data suggest that a peptide loop (residues 14-27) which shields the active site during catalysis is highly flexible in the free enzyme.

Asparaginase↗

PubMed1993

Ferricytochrome c induces monophasic kinetics of ferrocytochrome c oxidation in cytochrome c oxidase.

The kinetics of ferrocytochrome c oxidation by reconstituted cytochrome c oxidase (COX) from bovine heart was followed by a spectrophotometric method, using on-line data collection and subsequent calculation of reaction rates from a function fitted to the progress curve. When reaction rates were calculated at increasing reaction times, the multiphasic kinetics of ferrocytochrome c oxidation gradually changed into monophasic Michaelis-Menten kinetics. The same phenomenon was observed when ferrocytochrome c oxidation was followed in the presence of increasing amounts of ferricytochrome c. From these results we conclude that ferricytochrome c shifts the multiphasic kinetics of ferrocytochrome c oxidation by COX into monophasic kinetics, comparable to high ionic strength conditions. Furthermore, we show that ferricytochrome c inhibits the "high affinity phase" of ferrocytochrome c oxidation in an apparently competitive way, while inhibition of the "low affinity phase" is noncompetitive. These findings are consistent with a "regulatory site model" where both the catalytic and the regulatory site bind ferro- as well as ferricytochrome c.

Animals↗

PubMed1992

Site-specific mutagenesis of Escherichia coli asparaginase II. None of the three histidine residues is required for catalysis.

Site-specific mutagenesis was used to replace the three histidine residues of Escherichia coli asparaginase II (EcA2) with other amino acids. The following enzyme variants were studied: [H87A]EcA2, [H87L]EcA2, [H87K]EcA2, [H183L]EcA2 and [H197L]EcA2. None of the mutations substantially affected the Km for L-aspartic acid beta-hydroxamate or impaired aspartate binding. The relative activities towards L-Asn, L-Gln, and l-aspartic acid beta-hydroxamate were reduced to the same extent, with residual activities exceeding 10% of the wild-type values. These data do not support a number of previous reports suggesting that histidine residues are essential for catalysis. Spectroscopic characterization of the modified enzymes allowed the unequivocal assignment of the histidine resonances in 1H-NMR spectra of asparaginase II. A histidine signal previously shown to disappear upon aspartate binding is due to His183, not to the highly conserved His87. The fact that [H183L]EcA2 has normal activity but greatly reduced stability in the presence of urea suggests that His183 is important for the stabilization of the native asparaginase tetramer. 1H-NMR and fluorescence spectroscopy indicate that His87 is located in the interior of the protein, possibly adjacent to the active site.

Amino Acid Sequence↗

PubMed1992

Probing the role of threonine and serine residues of E. coli asparaginase II by site-specific mutagenesis.

Site-specific mutagenesis has been used to probe amino acid residues proposed to be critical in catalysis by Escherichia coli asparaginase II. Thr12 is conserved in all known asparaginases. The catalytic constant of a T12A mutant towards L-aspartic acid beta-hydroxamate was reduced to 0.04% of wild type activity, while its Km and stability against urea denaturation were unchanged. The mutant enzyme T12S exhibited almost normal activity but altered substrate specificity. Replacement of Thr119 with Ala led to a 90% decrease of activity without markedly affecting substrate binding. The mutant enzyme S122A showed normal catalytic function but impaired stability in urea solutions. These data indicate that the hydroxyl group of Thr12 is directly involved in catalysis, probably by favorably interacting with a transition state or intermediate. By contrast, Thr119 and Ser122, both putative target sites of the inactivator DONV, are functionally less important.

Amino Acid Sequence↗

PubMed1992

Cloning and sequence analyses of cDNAs encoding aminoacylase I from porcine kidney.

cDNAs encoding L-aminoacylase (EC 3.5.1.14) were isolated from a lambda gt10 cDNA library derived from porcine kidney mRNA. The clones were identified by hybridization with a synthetic oligonucleotide probe based on partial peptide sequences, or with a DNA probe encoding human aminoacylase I. Several cDNA clones isolated from the library had a length of about 1.3 kbp. They contained an open reading frame of 1218 bp encoding a polypeptide of 406 amino acids. The deduced amino-acid sequence contains the known peptide sequences; in addition, M(r) (45.3 kDa) and amino-acid composition of the predicted polypeptide match those of purified aminoacylase I. Data base searches did not reveal significant sequence homologies of aminoacylase I with other well-known amidases.

Amidohydrolases↗

PubMed1991

A catalytic role for threonine-12 of E. coli asparaginase II as established by site-directed mutagenesis.

A threonine-12 to alanine mutant of E. coli asparaginase II (EC 3.5.1.1) has less than 0.01% of the activity of wild-type enzyme. Both tertiary and quaternary structure of the enzyme are essentially unaffected by the mutation; thus the activity loss seems to be the result of a direct impairment of catalytic function. As aspartate is still bound by the mutant enzyme, Thr-12 appears not be involved in substrate binding.

Asparaginase↗

PubMed1990

Carbon-13 labelled biotin--a new probe for the study of enzyme catalyzed carboxylation and decarboxylation reactions.

[2'-13C]Biotin was incorporated into avidin (egg white), glutaconyl-CoA decarboxylase (EC 4.1.1.70) from Acidaminococcus fermentans and the biotin carrier of transcarboxylase from Propionibacterium freudenreichii (EC 2.1.3.1). 13C-NMR measurements showed an upfield shift of the carbonyl carbon of 3.1 and 2.0 ppm for both enzymes, whereas binding to avidin induced no significant change of the chemical shift as compared to free biotin. The data indicate that the enzymes provide an electronic environment for the covalently bound biotin which favours carboxylation. In addition it was demonstrated by NMR-measurements that glutaconyl-CoA decarboxylase, from which the hydrophobic carboxy-lyase subunit (beta) was removed, could carboxylate free biotin.

Avidin↗

PubMed1990

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.

Amidohydrolases↗

PubMed1989

On the role of histidine and tyrosine residues in E. coli asparaginase. Chemical modification and 1H-nuclear magnetic resonance studies.

The relative importance of tyrosine and histidine residues for the catalytic action of Escherichia coli asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) was studied by chemical modification and 1H-NMR spectroscopy. We show that, under appropriate reaction conditions, N-bromosuccinimide (NBS) as well as diazonium-1H-tetrazole (DHT) inactivate by selectively modifying two tyrosine residues per asparaginase subunit without affecting histidyl moieties. We further show that diethyl pyrocarbonate (DEP), a reagent considered specific for histidine, also modifies tyrosine residues in asparaginase. Thus, inactivation of the enzyme by DEP is not indicative of histidine residues being involved in catalysis. In 1H-nuclear magnetic resonance (NMR) spectra of asparaginase signals from all three histidine residues were identified. By measuring the pH dependencies of these resonances, pKa values of 7.0 and 5.8 were derived for two of the histidines. Titration with aspartate which tightly binds to the enzyme at low pH strongly reduced the signal amplitude of the pKa 7 histidyl moiety as well as those of resonances of one or more tyrosine residues. This suggests that tyrosine and histidine are indeed constituents of the active site.

Asparaginase↗

PubMed1989

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 degrees C and pH 7.0, the dissociation rate of the complex is approximately 6 x 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 microM. Molecular mechanics calculations showed that conformation and charge distribution of butylmalonate are strikingly similar to those of the putative transition state of aminoacylase catalysis.

Amidohydrolases↗

PubMed1989

Reactivities of sulfhydryl groups in native and metal-free aminoacylase I.

Aminoacylase I from porcine kidney (EC 3.5.1.14) contains seven cysteine residues per subunit. Three sulfhydryl groups are accessible to modification by 4-hydroxymercuribenzoate (p-MB). The kinetics of the reaction suggest that only one of these groups affects acylase activity when modified by p-MB. Its reaction rate increases 2-3-fold when the essential metal ion of aminoacylase is removed. Modification of metal-free apoenzyme by N-ethylmaleimide (NEM) abolishes its activity without impairing Zn2+ binding. This indicates that the sulfhydryl group reacting with NEM is not directly coordinated to the metal. DTNB (5,5'-Dithio-bis(2-nitrobenzoate), Ellman's reagent) also modifies three sulfhydryl groups per subunit. In this case, the reactivities of native aminoacylase and apoenzyme are not significantly different. N-Hydroxy-2-aminobutyrate, a strong aminoacylase inhibitor, substantially increases the reactivity of the slowest reacting sulfhydryl in both native enzyme and metal-free aminoacylase. It appears that binding of the inhibitor or removal of the metal ion induces conformational changes of the amino-acylase active site that render a buried sulfhydryl group more accessible to modification.

Amidohydrolases↗

PubMed1988

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.

Amidohydrolases↗

PubMed1988

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.

Amidohydrolases↗

PubMed1987

Unusual solvent isotope effects on the aminoacylase-catalyzed hydrolysis of acetylamino acids.

The deuterium solvent isotope effect on hydrolysis of acetylamino acids catalyzed by porcine kidney aminoacylase I (EC 3.5.1.14) was studied. With Ac-L-Met, a 'standard' aminoacylase substrate, the effect was normal at low pH (kcat(D)/kcat(H) = 0.7 at pH 6), virtually absent at neutrality, and distinctly inverse (kcat(D)/kcat(H) = 1.4) at pH 9. Km was not significantly affected. The rates of Ac-L-Phe hydrolysis in D2O considerably exceeded those in H2O at any pH between 6.5 and 9. We explain this unusual effect of D2O on aminoacylase I catalysis by an inverse equilibrium effect partially cancelling or, at high pH, reversing a normal isotope effect on kcat.

Amidohydrolases↗