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C Paech

Publications and source records attributed to C Paech.

At least 19 recordsLinked to original sources
PubMed2000

Benzophenone boronic acid photoaffinity labeling of subtilisin CMMs to probe altered specificity.

A transition state analogue inhibitor, boronic acid benzophenone (BBP) photoprobe, was used to study the differences in the topology of the S1 pocket of chemically modified mutant enzymes (CMMs). The BBP proved to be an effective competitive inhibitor and a revealing active site directed photoprobe of the CMMs of the serine protease subtilisin Bacillus lentus (SBL) which were chemically modified with the hydrophobic, negatively charged and positively charged moieties at the S1 pocket S166C residue. As expected, in all cases BBP bound best to WT-SBL. BBP binding to S166C-SCH2C6H5 and S166C-CH2-c-C6H11, with their large hydrophobic side chains, was reduced by 86-fold and 9-fold, respectively, compared to WT. Relative to WT, BBP binding to the charged CMMs, S166C-S-CH2CH2SO3- or S166C-S-CH2CH2NH3+, was reduced 170-fold and 4-fold respectively. Photolysis of the WT-SBL-BBP enzyme inhibitor (EI) complex, inactivated the enzyme and effected the formation of a covalent crosslink between WT and BBP. The crosslink was identified at Gly127 by peptide mapping analysis and Edman sequencing. Gly127 is located in the S1 hydrophobic pocket of SBL and its modification thus established binding of the benzophenone moiety in S1. Photolysis of the EI complex of S166C-SCH2C6H5, S166C-S-CH2CH2SO3-, or S166C-S-CH2CH2NH3+ and BBP under the same conditions did not inactivate these enzymes, nor effect the formation of a crosslink. These results corroborated the kinetic evidence that the active site topology of these CMMs is dramatically altered from that of WT. In contrast, while photolysis of the S166C-CH2-c-C6H11-BBP EI complex only inactivated 50% of the enzyme after 12 h, it still effected the formation of a covalent crosslink between the CMM and BBP, again at Gly127. However, this photolytic reaction was less efficient than with WT, demonstrating that the S1 pocket of S166C-CH2-c-C6H11 is significantly restricted compared to WT, but not as completely as for the other CMMs.

Amino Acid Sequence↗
PubMed1996

Active-site titration of serine proteases using a fluoride ion selective electrode and sulfonyl fluoride inhibitors.

We report a general procedure for the determination of active enzyme concentrations for serine proteases. The method relies on the measurement of fluoride ion released from sulfonyl fluorides upon reaction with the active-site serine using an ion selective electrode. The results have been independently confirmed by amino acid analyses of subtilisins and by spectrofluorometric and spectrophotometric titrations. The minimal enzyme concentration detectable is 1-10 microM protease. The method is insensitive to color and turbidity of the sample and is therefore useful for measuring protease concentration in broth solutions. The active enzyme concentration of subtilisin BPN' from Bacillus amyloliquefaciens determined by titration with phenylmethylsulfonyl fluoride is 25% higher than the concentration determined using the spectrophotometric burst titrant N-trans-cinnamoylimidazole. Analysis of the pre-steady-state burst amplitude and kinetics suggests that the extinction coefficient for the cinnamoyl acyl-enzyme is larger than previously measured and a significant fraction of the enzyme is present as an unproductive ES2 complex. The molar extinction coefficient at 280 nm for subtilisin BPN' is 26.5 mM-1 cm-1 and for subtilisin from Bacillus lentus is 22.5 mM-1 cm-1.

Bacillus↗
PubMed1996

Unusual ligand binding at the active site domain of an engineered mutant of subtilisin BL.

As an attempt to recruit the third calcium binding site of thermitase into subtilisin BL, a Bacillus lentus alkaline protease (BLAP), the amino acid sequence from position 50 to 60 and position 92 was modified to the equivalent amino acids in thermitase. The resulting protein, designated BLAPm109, exhibited unusual biochemical features. Peptide mapping and gel electrophoresis revealed that two protein species co-purify in a ratio of about 1:1. Form 1 consisted of a single polypeptide of 269 amino acid residues. Form 2 was the same protein but with an internal peptide bond cleavage at the C-terminus of position 54. On electropherograms a dimer of Form 1 and Form 2 was also detectable. A zymogram showed that all three molecular species were catalytically active. From this protein mixture, crystals suitable for X-ray analysis were nevertheless obtained. SDS-PAGE of protein recovered from a crystal revealed that only Form 2 appears. in the crystal. The space group for this crystal was P21 with unit cell dimensions of a=42 angstroms, b=58 angstroms, c=47 angstroms and beta = 106.3 degrees. Examination of the preliminary electron density map revealed that the "thermitase loop" from 50 to 60 departs from the surface of the protein and winds through the active site of a symmetry-related copy of the asymmetric unit.

Amino Acid Sequence↗
PubMed1994

Peptide mapping of subtilisins as a practical tool for locating protein sequence errors during extensive protein engineering projects.

Protein engineering based on rational design is an iterative process of sequential amino acid residue replacements. This requires a rapid and sensitive method for checking the protein sequence after each round of mutagenesis. As shown with subtilisin BL, acid treatment followed by urea denaturation renders the enzyme degradable by trypsin within 10 min. Separation of the peptides by reversed-phase HPLC produces a map that differentiates even the most conservative alteration on peptides as large as 48 amino acid residues. The method was used to uncover erroneous mutations; to determine the concentration of active protease relative to an internal standard of known specific activity; to measure the rate of oxidation of methionine-216 in the oxyanion hole of subtilisin BL; and to document that under these conditions no other methionine in the molecule is oxidized by hydrogen peroxide.

Amino Acid Sequence↗
PubMed1993

Zymogram of proteases made with developed film from nondenaturing polyacrylamide gels after electrophoresis.

A simple, extremely versatile method for preparing zymograms from proteases after nondenaturing Phast-System gel electrophoresis is described. After completion of the run and before staining, an electropherogram and a piece of developed, single-side coated X-ray film are brought into contact for 5 min at room temperature. The film overlay is then separated and the gel is stained for protein. The zymogram on the X-ray film is generated by simply pouring about 10 ml of a suitable buffer solution at 30 to 50 degrees C over the film strip. Clearing zones appeared within a few seconds to a few minutes depending on protease amount. For the alkaline protease subtilisin BL the lower limit of detectability was 10 ng applied to the gel prior to electrophoresis. For preservation and archiving the zymogram film is simply rinsed with distilled water and air-dried. The film can be cut to size and mounted for a slide projector, or the film can serve as a negative for photographic enlargements. The clearing zone area is proportional to the amount of protease from 10 to 100 ng of protein.

Bacillus subtilis↗
PubMed1993

Strategy and implementation of a system for protein engineering.

This paper describes an overall view of an industrial protein engineering project from conception to successful completion. The choice of rational design was determined by the availability of an excellent three-dimensional crystal structure and the availability of information in the literature to define a strategy. The design strategy was refined extensively during the course of the project. The development of methods for mutagenesis, expression, verification, purification, and characterization of mutant enzymes is dictated in part by the enzyme property one chooses to modify and must be rapid yet accurate. Such an approach would be applicable to improve the stability of any other protein or enzyme. Using this approach, we successfully increased the stability of subtilisin BL over 10-fold at 50 degrees C with an overall success rate greater than 60%.

Bacillus↗
PubMed1992

The crystal structure of the Bacillus lentus alkaline protease, subtilisin BL, at 1.4 A resolution.

The crystal structure of subtilisin BL, an alkaline protease from Bacillus lentus with activity at pH 11, has been determined to 1.4 A resolution. The structure was solved by molecular replacement starting with the 2.1 A structure of subtilisin BPN' followed by molecular dynamics refinement using X-PLOR. A final crystallographic R-factor of 19% overall was obtained. The enzyme possesses stability at high pH, which is a result of the high pI of the protein. Almost all of the acidic side-chains are involved in some type of electrostatic interaction (ion pairs, calcium binding, etc.). Furthermore, three of seven tyrosine residues have potential partners for forming salt bridges. All of the potential partners are arginine with a pK around 12. Lysine would not function well in a salt bridge with tyrosine as it deprotonates at around the same pH as tyrosine ionizes. Stability at high pH is acquired in part from the pI of the protein, but also from the formation of salt bridges (which would affect the pI). The overall structure of the enzyme is very similar to other subtilisins and shows that the subtilisin fold is more highly conserved than would be expected from the differences in amino acid sequence. The amino acid side-chains in the hydrophobic core are not conserved, though the inter-residue interactions are. Finally, one third of the serine side-chains in the protein have multiple conformations. This presents an opportunity to correlate computer simulations with observed occupancies in the crystal structure.

Amino Acid Sequence↗
PubMed1987

Free amino Acid content and metabolic activities of setting and aborting soybean ovaries.

Fruits of soybean (Glycine max [L.] Merr.) that are destined to abscise shortly after anthesis grow more slowly than fruits that will be retained. In this work, amino acid composition, protein metabolism, and nucleic acid metabolism were studied in setting and abscising soybean ovaries from anthesis to 6 days after anthesis to provide additional evidence of chemical processes associated with abscission. Principal free amino acids were asparagine, aspartic acid, glutamic acid, serine, and glutamine. Percent aspartate and glutamate declined as the ovaries grew, with aspartate declining more in abscising and glutamate more in setting ovaries. Percent glutamate was positively correlated to percent abscission throughout the period. Proline, serine, and leucine were positively correlated to abscission from 0 to 2 days after anthesis, whereas significant negative correlations were observed at these ages for ethanolamine and arginine. (75)Se fed as selenate and (14)C fed as sucrose, glycine, and alanine were readily incorporated into soluble and insoluble proteins in a 24-hour in vitro incubation. Radioactivity of total proteins, expressed on a perovary basis, was negatively correlated with percent abscission and positively correlated with ovary weight. [(14)C]Glutamine and serine followed the opposite pattern, with greater protein labeling in abscising than in setting ovaries. When data were expressed as disintegrations per minute per milligram ovary fresh weight, protein labeling from alanine was seen to be significantly greater in abscising ovaries at anthesis and throughout the sampling period. Nucleic acid labeling from uridine was highly correlated to ovary weight; labeling from thymidine was greater in setting than abscising ovaries at anthesis and in abscising ovaries at later stages of development. It is concluded that abscising ovaries can continue amino acid metabolism almost up to the date of separation from the raceme, and that the involvement of alanine, glutamine, aspartate, glutamate, and other amino acids in soybean flower abortion deserves further study.

Journal Article↗
PubMed1986

Kinetics of ribulosebisphosphate carboxylase at high protein concentration.

Kinetic parameters of ribulos-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase) are usually evaluated in dilute solutions (less than 0.1 mg ml-1). Yet, this enzyme occurs in vivo at 100-200 mg ml-1 and a total protein concentration 300-400 mg ml-1. Enzymes can change their catalytic properties upon 'crowding'. Hence it became of interest to determine whether RuBP carboxylase elicits any properties not observable in dilute solution. Pre-steady state progress curves of fully activated enzyme showed an initial burst followed by a slower rate of product formation. The extent of the burst increased as concentration ratios of RuBP and RuBP carboxylase decreased. The burst corresponds to 1/8 turnover per holoenzyme or 1 turnover per active site. No discontinuity in progress curves was observed with partially activated enzyme.

Chloroplasts↗
PubMed1986

Purification and degradation of ribulose bisphosphate carboxylase from soybean leaves.

A rapid method is described for the preparation of up to 500 milligrams of pure ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase) from 250 grams of field-grown soybean leaves. Leaves were extracted in 20 millimolar phosphate (pH 6.9) at 4 degrees C, containing 4% (w/v) polyvinylpolypyrrolidone, 10 micromolar leupeptin, 1 millimolar phenylmethyl sulfonylfluoride, 1 millimolar diethyldithiocarbamate, 5 millimolar MgCl(2), 1 millimolar dithiothreitol, 0.2 millimolar ethylene-diaminetetraacetic acid, 50 millimolar 2-mercaptoethanol. The extract was incubated in the presence of 5 millimolar ATP at 58 degrees C for 9 minutes, then centrifuged and concentrated. Sucrose gradient centrifugation into 8 to 28% (w/v) sucrose on a vertical rotor for 2.5 hours yielded pure enzyme with a specific activity of 1.1 to 1.3 micromoles per minute per milligram protein at pH 8.0, 25 degrees C. Soybean plants of the same line grown (at 400 microeinsteins per square meter per second) in growth chambers yielded enzyme with a specific activity of 0.6 to 0.7 micromoles per minute per milligram protein. During prolonged purification procedures a proteolytic degradation of RuBP carboxylase caused complete loss of catalytic activity. Without destroying the quaternary structure of the enzyme, a 3 kilodalton peptide was removed from all large subunits before further breakdown (removal of a 5 kilodalton peptide) occurred. Catalytic competence of the enzyme was abolished with the loss of the first (3 kilodalton) peptide.

Journal Article↗
PubMed1986

Biochemical characterization of soybean ovary growth from anthesis to abscission of aborting ovaries.

Soybean (Glycine max [L.] Merr.) ovary growth was measured from anthesis to 6 days after anthesis (DAA) to establish a timetable of biochemical events that might be useful in identifying processes that initiate abscission. Two procedures were developed to provide samples with either high or low percent pod set for ;IX93-100,' a semideterminate line having long racemes. Characteristics measured were fresh and dry weight, soluble and insoluble protein, soluble carbohydrate, starch, RNA, and DNA. Setting ovaries grew more rapidly than abscising ovaries. Since there was a daily increase in ovary weight in both groups, all measured characteristics showed daily increases when expressed on perovary basis. Statistically significant differences between groups were detected between 2 and 5 DAA for most characteristics. When chemical composition was expressed on concentration basis, starch level was significantly higher in setting ovaries at 5 and 6 DAA. Regression analysis showed that these deviations between setting and abscising samples started between anthesis and 1 DAA. We conclude that processes leading to eventual shedding of fertilized ovaries (called flower abortion in soybeans) commence soon after anthesis of the shed flower, and that setting and abscising ovaries do not differ in protein, soluble carbohydrate, starch, or nucleic acid content when abscission processes begin.

Journal Article↗
PubMed1982

Simplified isolation and molecular composition of NADH dehydrogenase of the respiratory chain.

A simplified procedure for the isolation of NADH dehydrogenase from the inner membrane of ox heart mitochondria is presented which permits relatively rapid preparation of the enzyme in a more stable form than that afforded by published methods. The protein thus isolated displays more than eight different subunits in gel electrophoresis under denaturing conditions, three of which are also present in the "low-molecular-weight form' of the enzyme prepared under more drastic conditions. Complex I contains several subunits, mostly of low molecular weight, not seen in soluble purified NADH dehydrogenase. It is suggested that some of these may be 'binding peptides' necessary in linking NADH dehydrogenase to ubiquinone reduction, analogously to the role of small peptides in linking succinate dehydrogenase to ubiquinone. The dehydrogenase isolated by the rapid method contains equimolar amounts of non-haem iron and labile sulphur, but on further manipulation non-haem iron (but no labile sulphur) is lost, resulting in ratios of S/Fe in excess of unity, as previously reported for preparations isolated by longer procedures.

Animals↗
PubMed1981

Structural identification of iron-sulfur clusters of the respiratory chain-linked NADH dehydrogenase.

The structural nature of the iron-sulfur clusters of NADH dehydrogenase from beef heart mitochondria has been studied by the cluster extrusion technique. Enzyme samples were unfolded anaerobically in 80% (v/v) hexamethylphosphoramide/aqueous buffer in the presence of o-xylyl-alpha,alpha'-dithiol as the displacing agent and the extruded clusters were then reacted with p-trifluoromethylbenzenethiol and analyzed by Fourier transform 19F NMR at 339 MHz. Whenever extrusion was nearly complete, both binuclear and tetranuclear clusters were found at a mole ratio of approximately 2:1. Thus, the dehydrogenase, with 16 g atoms of non-heme iron present/mol of FMN, contains most likely four [2Fe-2S] and two [4Fe-4S] clusters. Because the enzyme contains four or, at the most five, EPR-detectable iron-sulfur centers, it appears that one or more of the clusters are EPR-silent.

Anaerobiosis↗
PubMed1978

Active site studies of ribulose-1,5-bisphosphate carboxylase/oxygenase with pyridoxal 5'-phosphate.

There are 16 epsilon-amino groups of lysyl residues which are essential for the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. These lysyl residues formed a Schiff base with pyridoxal 5'-phosphate which was stabilized by NaBH4 reduction. The stoichiometry of covalently bound pyridoxal 5'-phosphate after NaBH4 reduction was determined spectrophotometrically with a derived molar extinction coefficient of 4800 M-1 cm-1. The incorporation of pyridoxal 5'-phosphate into the protein was accompanied by loss of the carboxylase and oxygenase activities, but the ratio of their activities remained constant. Vmax, but not Km, values were changed by this modification of the amino acid groups. Half of the epsilon-amino groups of lysine appeared to be at the 8 catalytic sites and half at the 8 activator sites for CO2, as indicated by kinetics of reactivation of the enzyme activity during dissociation of the Schiff base between pyridoxal 5'-phosphate and the protein. Reduction with NaB3H4 revealed that all 16 of the lysyl residues were on the large subunit. Ribulose-1,5-bis-phosphate alone protected 16 amino groups from Schiff base formation with pyridoxal 5'-phosphate, and the enzyme activity was fully conserved. NaHCO3 increased and MgCl2 lowered slightly the protective effect of ribulose-1,5-bisphosphate. Modification of sulfhydryl groups by p-chloromercuribenzoic acid inhibited the enzyme and excluded binding of 8 equivalents of pyridoxal 5'-phosphate. Upon removal of the mercuribenzoate groups with excess dithiothreitol, the loss of enzyme activity was exponentially correlated with the binding of 8 mol of pyridoxal 5'-phosphate/mol of enzyme. In contrast to p-chloromercuribenzoic acid, iodoacetamide, which inhibited the catalysis, had no influence on the binding of the 16 pyridoxal 5'-phosphate/mol of enzyme. It is suggested that the CO2 activator site with one epsilon-amino group for binding of CO2 and the catalytic site with one epsilon-amino group of a yet unknown function are located closely together on the large subunits of the enzyme. The results are consistent with a sulfhydryl group as the proton acceptor opposite carbon 3 of ribulose-1,5-bisphosphate, and when this sulfhydryl group is blocked by the bulky mercuribenzoate group, but not by the small carboxyamidomethyl group, pyridoxal 5'-phosphate binding is modified.

Binding Sites↗