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R Rink

Publications and source records attributed to R Rink.

At least 19 recordsLinked to original sources
PubMed2004

Probing the self-assembly and the accompanying structural changes of hydrophobin SC3 on a hydrophobic surface by mass spectrometry.

The fungal class I hydrophobin SC3 self-assembles into an amphipathic membrane at hydrophilic-hydrophobic interfaces such as the water-air and water-Teflon interface. During self-assembly, the water-soluble state of SC3 proceeds via the intermediate alpha-helical state to the stable end form called the beta-sheet state. Self-assembly of the hydrophobin at the Teflon surface is arrested in the alpha-helical state. The beta-sheet state can be induced at elevated temperature in the presence of detergent. The structural changes of SC3 were monitored by various mass spectrometry techniques. We show that the so-called second loop of SC3 (C39-S72) has a high affinity for Teflon. Binding of this part of SC3 to Teflon was accompanied by the formation of alpha-helical structure and resulted in low solvent accessibility. The solvent-protected region of the second loop extended upon conversion to the beta-sheet state. In contrast, the C-terminal part of SC3 became more exposed to the solvent. The results indicate that the second loop of class I hydrophobins plays a pivotal role in self-assembly at the hydrophilic-hydrophobic interface. Of interest, this loop is much smaller in case of class II hydrophobins, which may explain the differences in their assembly.

Air↗
PubMed2003

Massive magnetic-field-induced structural transformation in Gd5Ge4 and the nature of the giant magnetocaloric effect.

A massive magnetic-field-induced structural transformation in Gd5Ge4, which occurs below 30 K, was imaged at the atomic level by uniquely coupling high-resolution x-ray powder diffraction with magnetic fields up to 35 kOe. In addition to uncovering the nature of the magnetic field induced structural transition, our data demonstrate that the giant magnetocaloric effect, observed in low magnetic fields, arises from the amplification of a conventional magnetic entropy-driven mechanism by the difference in the entropies of two phases, borne by the concomitant structural transformation.

Journal Article↗
PubMed2000

Tyrosine residues serve as proton donor in the catalytic mechanism of epoxide hydrolase from Agrobacterium radiobacter.

Epoxide hydrolase from Agrobacterium radiobacter catalyzes the hydrolysis of epoxides to their diols via an alkyl-enzyme intermediate. The recently solved X-ray structure of the enzyme shows that two tyrosine residues (Tyr152 and Tyr215) are positioned close to the nucleophile Asp107 in such a way that they can serve as proton donor in the alkylation reaction step. The role of these tyrosines, which are conserved in other epoxide hydrolases, was studied by site-directed mutagenesis. Mutation of Tyr215 to Phe and Ala and mutation of Tyr152 to Phe resulted in mutant enzymes of which the k(cat) values were only 2-4-fold lower than for wild-type enzyme, whereas the K(m) values for the (R)-enantiomers of styrene oxide and p-nitrostyrene oxide were 3 orders of magnitude higher than the K(m) values of wild-type enzyme, showing that the alkylation half-reaction is severely affected by the mutations. Pre-steady-state analysis of the conversion of (R)-styrene oxide by the Y215F and Y215A mutants showed that the 1000-fold elevated K(m) values were mainly caused by a 15-40-fold increase in K(S) and a 20-fold reduction in the rate of alkylation. The rates of hydrolysis of the alkyl-enzyme intermediates were not significantly affected by the mutations. The double mutant Y152F+Y215F showed only a low residual activity for (R)-styrene oxide, with a k(cat)/K(m) value that was 6 orders of magnitude lower than with wild-type enzyme and 3 orders of magnitude lower than with the single tyrosine mutants. This indicates that the effects of the mutations were cumulative. The side chain of Gln134 is positioned in the active site of the X-ray structure of epoxide hydrolase. Mutation of Gln134 to Ala resulted in an active enzyme with slightly altered steady-state kinetic parameters compared to wild-type enzyme, indicating that Gln134 is not essential for catalysis and that the side chain of Gln134 mimics bound substrate. Based upon this observation, the inhibitory potential of various unsubstituted amides was tested, resulting in the identification of phenylacetamide as a competitive inhibitor with an inhibition constant of 30 microM.

Acetamides↗
PubMed1999

The x-ray structure of epoxide hydrolase from Agrobacterium radiobacter AD1. An enzyme to detoxify harmful epoxides.

Epoxide hydrolases catalyze the cofactor-independent hydrolysis of reactive and toxic epoxides. They play an essential role in the detoxification of various xenobiotics in higher organisms and in the bacterial degradation of several environmental pollutants. The first x-ray structure of one of these, from Agrobacterium radiobacter AD1, has been determined by isomorphous replacement at 2.1-A resolution. The enzyme shows a two-domain structure with the core having the alpha/beta hydrolase-fold topology. The catalytic residues, Asp107 and His275, are located in a predominantly hydrophobic environment between the two domains. A tunnel connects the back of the active-site cavity with the surface of the enzyme and provides access to the active site for the catalytic water molecule, which in the crystal structure, has been found at hydrogen bond distance to His275. Because of a crystallographic contact, the active site has become accessible for the Gln134 side chain, which occupies a position mimicking a bound substrate. The structure suggests Tyr152/Tyr215 as the residues involved in substrate binding, stabilization of the transition state, and possibly protonation of the epoxide oxygen.

Amino Acid Sequence↗
PubMed1998

Kinetic mechanism of the enantioselective conversion of styrene oxide by epoxide hydrolase from Agrobacterium radiobacter AD1.

Epoxide hydrolase from Agrobacterium radiobacter AD1 catalyzes the enantioselective hydrolysis of styrene oxide with an E value of 16. The (R)-enantiomer of styrene oxide is first converted with a k(cat) of 3.8 s(-1), and the conversion of the (S)-enantiomer is inhibited. The latter is subsequently hydrolyzed with a k(cat) of 10.5 s(-1). The pre-steady-state kinetic parameters were determined for both enantiomers with stopped-flow fluorescence and rapid-quench techniques. For (R)-styrene oxide a four-step mechanism was needed to describe the data. It involved the formation of a Michaelis complex that is in rapid equilibrium with free enzyme and substrate, followed by rapid and reversible alkylation of the enzyme. A unimolecular isomerization of the alkylated enzyme precedes the hydrolysis of the covalent intermediate, which could be observed due to an enhancement of the intrinsic protein fluorescence during this step. The conversion of (S)-styrene oxide could be described by a three-step mechanism, which also involved reversible and rapid formation of an ester intermediate from a Michaelis complex and its subsequent slow hydrolysis as the rate-limiting step. The unimolecular isomerization step has not been observed for rat microsomal epoxide hydrolase, for which a kinetic mechanism was recently established [Tzeng, H.-F., Laughlin, L. T., Lin, S., and Armstrong, R. N. (1996) J. Am. Chem. Soc. 118, 9436-9437]. For both enantiomers of styrene oxide, the Km value was much lower than the substrate binding constant K(S) due to extensive accumulation of the covalent intermediate. The enantioselectivity was more pronounced in the alkylation rates than in the rate-limiting hydrolysis steps. The combined reaction schemes for (R)- and (S)-styrene oxide gave an accurate description of the epoxide hydrolase catalyzed kinetic resolution of racemic styrene oxide.

Binding Sites↗
PubMed1997

Primary structure and catalytic mechanism of the epoxide hydrolase from Agrobacterium radiobacter AD1.

The epoxide hydrolase gene from Agrobacterium radiobacter AD1, a bacterium that is able to grow on epichlorohydrin as the sole carbon source, was cloned by means of the polymerase chain reaction with two degenerate primers based on the N-terminal and C-terminal sequences of the enzyme. The epoxide hydrolase gene coded for a protein of 294 amino acids with a molecular mass of 34 kDa. An identical epoxide hydrolase gene was cloned from chromosomal DNA of the closely related strain A. radiobacter CFZ11. The recombinant epoxide hydrolase was expressed up to 40% of the total cellular protein content in Escherichia coli BL21(DE3) and the purified enzyme had a kcat of 21 s-1 with epichlorohydrin. Amino acid sequence similarity of the epoxide hydrolase with eukaryotic epoxide hydrolases, haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, and bromoperoxidase A2 from Streptomyces aureofaciens indicated that it belonged to the alpha/beta-hydrolase fold family. This conclusion was supported by secondary structure predictions and analysis of the secondary structure with circular dichroism spectroscopy. The catalytic triad residues of epoxide hydrolase are proposed to be Asp107, His275, and Asp246. Replacement of these residues to Ala/Glu, Arg/Gln, and Ala, respectively, resulted in a dramatic loss of activity for epichlorohydrin. The reaction mechanism of epoxide hydrolase proceeds via a covalently bound ester intermediate, as was shown by single turnover experiments with the His275 --> Arg mutant of epoxide hydrolase in which the ester intermediate could be trapped.

Amino Acid Sequence↗
PubMed1997

Partial nephrectomy for pediatric renal cell carcinoma: an unusual case presentation.

Nine years after successful treatment of Stage IV neuroblastoma, a 10-year-old white girl was demonstrated to have a complex cystic mass in the upper pole of her solitary right kidney. Partial nephrectomy was performed, disclosing a renal cell carcinoma, predominantly clear cell type. No metastases were detected. Renal cell carcinoma is a rare cause of secondary malignancy. Partial nephrectomy can be used successfully to treat renal cell carcinoma in children.

Carcinoma, Renal Cell↗
PubMed1997

A buccal mucosal harvesting technique for urethral reconstruction.

PURPOSE: A step-by-step harvesting technique for buccal mucosa is described that maximizes graft yield while minimizing potential donor site morbidity in urethral reconstruction. MATERIALS AND METHODS: A specialized oral retractor was used to expose and retract the buccal mucosal lining of the oral cavity for graft harvesting in 12 patients. RESULTS: Adequate buccal mucosal graft size was obtained for each reconstruction. There were no oral donor site or urethral recipient site complications. CONCLUSIONS: This harvesting technique offers a simple and effective method for optimizing buccal mucosal graft harvests.

Adult↗
PubMed1996

Kinetic characterization and X-ray structure of a mutant of haloalkane dehalogenase with higher catalytic activity and modified substrate range.

Conversion of halogenated aliphatics by haloalkane dehalogenase proceeds via the formation of a covalent alkyl-enzyme intermediate which is subsequently hydrolyzed by water. In the wild type enzyme, the slowest step for both 1,2-dichloroethane and 1,2-dibromoethane conversion is a unimolecular enzyme isomerization preceding rapid halide dissociation. Phenylalanine 172 is located in a helix-loop-helix structure that covers the active site cavity of the enzyme, interacts with the C1 beta of 1,2-dichloroethane during catalysis, and could be involved in stabilization of this helix-loop-helix region of the cap domain of the enzyme. To obtain more information about the role of this residue in dehalogenase function, we performed a mutational analysis of position 172 and studied the kinetics and X-ray structure of the Phe172Trp enzyme. The Phe172Trp mutant had a 10-fold higher Kcat/Km for 1-chlorohexane and a 2-fold higher Kcat for 1,2-dibromoethane than the wild-type enzyme. The X-ray structure of the Phe172Trp enzyme showed a local conformational change in the helix-loop-helix region that covers the active site. This could explain the elevated activity for 1-chlorohexane of the Phe172Trp enzyme, since it allows this large substrate to bind more easily in the active site cavity. Pre-steady-state kinetic analysis showed that the increase in Kcat found for 1,2-dibromoethane conversion could be attributed to an increase in the rate of an enzyme isomerization step that preceeds halide release. The observed conformational difference between the helix-loop-helix structures of the wild-type enzyme and the faster mutant suggests that the isomerization required for halide release could be a conformational change that takes place in this region of the cap domain of the dehalogenase. It is proposed that Phe172 is involved in stabilization of the helix-loop-helix structure that covers the active site of the enzyme and creates a rigid hydrophobic cavity for small apolar halogenated alkanes.

Amino Acids↗
PubMed1993

Construction of an expression and site-directed mutagenesis system of haloalkane dehalogenase in Escherichia coli.

Haloalkane dehalogenase from Xanthobacter autotrophicus was efficiently expressed in Escherichia coli BL21 (DE3) and E. coli JM101. After introduction of restriction sites by PCR the haloalkane dehalogenase gene (dhlA) was translationally fused behind the T7 (phi 10), trc, and tac promoters. This resulted in expression at 30 degrees C up to 38 and 18% of the total soluble cellular protein with the T7 and trc promoters, respectively. Dehalogenase expression under control of the tac promoter was below 1% of the soluble cell protein, however. Aggregation of haloalkane dehalogenase into inclusion bodies was found during growth at 37 degrees C but not at 30 degrees C. Aggregates were also formed from intact enzyme upon incubation at 37 degrees C of cells or crude extracts containing active mature dehalogenase. The high level of expression resulted in a short purification procedure in which 30-35 mg highly enriched haloalkane dehalogenase was obtained from an 0.51 culture. For the production of single-stranded DNA an f1(+) origin was introduced in the T7 expression system.

Amino Acid Sequence↗
PubMed1990

An enzymatic assay for the colorimetric and fluorimetric determination of uric acid in sera.

The methods described in this paper are based on the uricase catalyzed oxidation of uric acid to allantoine and hydrogen peroxide. By making use of the catalytic activity of peroxidase the generated H2O2 is measured either spectrophotometrically with 3-methyl-benzothiazoline-2-one hydrazone (MBTH) and 3-dimethylaminobenzoic acid (DMAB) (M1) or fluorimetrically with tyramine (M2) or L-tyrosine (M3). The methods are simple, sensitive and selective. The procedures developed can be rapidly and readily performed on patient serum samples without deproteinization using 100 microliters and 5 microliters for colorimetric and fluorimetric assay, respectively.

Colorimetry↗
PubMed1990

Plasma albumin repletion after transfusion with polymerized hemoglobin.

Pyridoxalated polymerized hemoglobin (PPHG) has promise as a blood substitute for transfusing patients with hemorrhage. Exchange transfusion with PPHG depletes plasma proteins. The purpose of this study was to determine if, during the early repletion of intravascular proteins, albumin was transported from the interstitium of skin or skeletal muscle into the vascular compartment. PPHG was prepared from stroma-free human hemoglobin (100-120 mg/ml). The Hct of anesthetized rats dropped from 42 +/- 4% to 10 +/- 1% after exchange transfusion. Immediately postexchange plasma albumin declined from 24 +/- 2 to 6 +/- 3 mg/ml. Five hours postexchange transfusion plasma albumin had doubled and the skin and skeletal muscle albumin content was 80% of control. These data indicate that a shift of interstitial albumin from skin and skeletal muscle can rapidly replace plasma protein deficits after massive transfusion with PPHG.

Animals↗
PubMed1987

Exchange-transfusion with pyridoxalated polymerized hemoglobin: effects on liver PO2.

To examine the efficacy of modified stroma-free hemoglobin in maintaining liver PO2, rats were exchange-transfused to hematocrit 10% using pyridoxalated polymerized hemoglobin (plp-polyHb, 10-12 g/dl) prepared from crystalline Hb. Following hemodilution, plasma Hb was 7.4 g/dl, and rats were normotensive. Mean liver PO2 was 3.4 vs 23.3 mm Hg in sham-exchanged controls. Other rats, hemodiluted similarly with 6% albumin or hydroxyethylstarch, were hypotensive and died. At 24 hours plasma Hb was 2.0 g/dl, indicating an intravascular half-life of approximately 16 hours. Hepatic PO2 was 12.4 vs 26.8 mm Hg in nonhemodiluted controls. Data provided by clearance of low-dose indocyanine green suggested reduced plasma volume and depressed liver blood flow. Scattered foci of midzonal hypoxic damage were observed in liver lobules. The basis for hypoxic injury is considered to be due in part to the acute restriction of oxygen supply induced by exchange-transfusion with plp-polyHb. The rate of loss of intravascular hemoglobin and diminished plasma volume could have contributed to oxygen insufficiency as well. Endotoxin present in the plp-polyHb was not a factor.

Animals↗