Gunter Fischer

The Escherichia coli SlyD Is a Metal Ion-regulated Peptidyl-prolyl cis/trans-Isomerase*

In Escherichia coli as many as nine different genes coding for proteins with significant homology to peptidyl-prolyl cis/trans-isomerases (PPIases) have been found. However, for three of them, the histidine-rich SlyD, the homologous gene product of ORF149, and parvulin-like SurA, it was not known whether these proteins really possess PPIase activity. To gain access to the full set of PPIases in E. coli, SlyD, the N-terminal fragment of SlyD devoid of the histidine-rich region, as well as the protein product of ORF149 of E. coli named SlpA (SlyD-like protein) were cloned, overexpressed, and purified to apparent homogeneity. On the basis of the amino acid sequences, both proteins proved to be of the FK506-binding protein type of PPIases. Only when using trypsin instead of chymotrypsin as helper enzyme in the PPIase assay, the enzymatic activity of full-length SlyD and its N-terminal fragment can be measured. For Suc-Ala-Phe-Pro-Arg-4-nitroanilide as substrate,k cat/K m of 29,600m −1 s−1 for SlyD and 18,600m −1 s−1 for the N-terminal fragment were obtained. Surprisingly, the PPIase activity of SlyD is reversibly regulated by binding of three Ni2+ ions to the histidine-rich, C-terminal region. Because the PPIase activity of SlpA could be established as well, we now know eight distinct PPIases with proven enzyme activity in E. coli.

Conformational specificity of chymotrypsin toward proline-containing substrates

A number of peptide-4-nitroanilide substrates containing proline within the peptide chain have been synthesized and subjected to chymotryptic hydrolysis. Values of kcat and Km have been obtained from measurements at pH 7.8 and 25.0 degrees C. Kinetic studies at high enzyme concentrations up to 6.0 X 10(-4) mol X 1(-1) have allowed the evaluation of the conformational specificity of chymotrypsin due to the observation of various kinetic phases during the time-course of the reaction. When proline occupies the P2 position within the peptide chain, it is shown that the enzyme cleaves only the trans isomer of the substrate. The conformational specificity has also been studied for proline in P4 and P5 positions of the substrate. In some cases, an enzyme-catalyzed hydrolysis of the cis isomer was detected. From the amplitude ratios and the rate constants of the kinetic phases, information about the structural dependency of the cis/trans interconversion could be obtained. Charged residues N-terminal to the isomeric bond are of little influence on either cis/trans ratio or the rate of cis to trans interconversion. Extending the peptide chain N-terminal to the isomeric bond by alanine decreases to a low extent the cis content and increases the rate constant of the trans isomer formation.

The refolding of urea-denatured ribonuclease A is catalyzed by peptidyl-prolyl cis-trans isomerase

The refolding of urea-denatured ribonuclease A was measured at 0.31-3.1 mol . l-1 urea in the presence of various concentrations of peptidyl-prolyl cis-trans isomerase isolated from pig kidney. The rate of the slow CT-phase in the refolding reaction was found to be sensitive to this enzyme. A rate enhancement proportional to the isomerase activity has been observed. The activity of the enzyme was assayed with Glt-Ala-Ala-Pro-Phe-4-nitroanilide. The catalytic activity of the isomerase against unfolded ribonuclease is suppressed after preincubation of the enzyme with 0.001 mol . l-1 Cu2+, 0.01 mol . l-1 H+ and by heat inactivation. The results indicate the involvement of the cis/trans interconversion of proline peptide bonds during the refolding of ribonuclease A.

The conformation around the peptide bond between the P1- and P2-positions is important for catalytic activity of some proline-specific proteases.

Proline-containing dipeptidyl-4-nitroanilides have been synthesised and subjected to dipeptidyl peptidase IV-catalysed hydrolysis at high enzyme concentrations to collect information on the conformational specificity of the enzyme active site for a nonscissile bond. Descriptions of the biphasic kinetics were carried out in terms of cis/trans interconversion of the substrates. The results show that the enzyme can cleave only the trans-conformation of the substrate. The competitive inhibition by Gly-Pro-OH and Ala-Pro-OH is also specific for the trans form of the dipeptides. The interpretation of the results obtained from these kinetic studies has led to proposals for the stepwise cleavage of biologically active peptides like substance P and beta-casomorphine by dipeptidyl peptidase IV.

Kinetic β‐deuterium isotope effects suggest a covalent mechanism for the protein folding enzyme peptidylprolyl cis/trans‐isomerase

The cis/trans interconversion of Glt‐Ala‐Ala‐Pro‐Phe‐4‐nitroanilide and Glt‐Ala‐Gly‐Pro‐Phe‐4‐nitroanilide was studied both enzymatically and nonenzymatically by measuring kinetic β‐deuterium isotope effects. The hydrogen atom at the α‐carbon atom of the Xaa residue within the Xaa‐Pro moiety was substituted by deuterium. In the nonenzymatic case the transition state of rotation is reflected by k H/k D > 1. When catalysed by 17 kDa PPIase the same bond rotation is characterized by k H/k D < 1. This suggests a covalent mechanism of catalysis which involves an approximately tetravalent carbon of the prolyl imidic bond for the transition state of reaction.

Functional Replacement of the Essential ESS1 in Yeast by the Plant Parvulin DlPar13

A functionally Pin1-like peptidyl-prolylcis/trans isomerase (PPIase1) was isolated from proembryogenic masses (PEMs) of Digitalis lanata according to its enzymatic activity. Partial sequence analysis of the purified enzyme (DlPar13) revealed sequence homology to members of the parvulin family of PPIases. Similar to human Pin1 and yeast Ess1, it exhibits catalytic activity toward substrates containing (Thr(P)/Ser(P))-Pro peptide bonds and comparable inhibition kinetics with juglone. Unlike Pin1-type enzymes it lacks the phosphoserine or phosphothreonine binding WW domain. Western blotting with anti-DlPar13 serum recognized the endogenous form in nucleic and cytosolic fractions of the plant cells. Since thePIN1 homologue ESS1 is an essential gene, complementation experiments in yeast were performed. When overexpressed in Saccharomyces cerevisiae DlPar13 is almost as effective as hPin1 in rescuing the temperature-sensitive phenotype caused by a mutation in ESS1. In contrast, the human parvulin hPar14 is not able to rescue the lethal phenotype of this yeast strain at nonpermissive temperatures. These results suggest a function for DlPar13 rather similar to parvulins of the Pin1-type.

Dipeptidylpeptidase IV--inactivation with N-peptidyl-O-aroyl hydroxylamines.

Eleven N-peptidyl-O-aroyl hydroxylamines have been synthesized and their hydrolytic stability, acidity and properties during reaction with dipeptidyl peptidase IV (E.C. 3.4.14.5) investigated. N-peptidyl-O-(4-nitrobenzoyl) hydroxylamines act as irreversible inhibitors of serine proteases. The serine enzyme, dipeptidyl peptidase IV (DP IV), is inactivated by substrate analog derivatives of this class by a suicide inactivation mechanism. During the enzyme reaction of DP IV with the suicide substrates most molecules are hydrolyzed but some irreversibly inactivate the target enzyme. In contrast to porcine pancreatic elastase and thermitase, DP IV exhibits a high ratio for hydrolysis of the compounds versus inhibition during their interaction with the enzyme. Variation of the leaving aroyl residue lowers this ratio. Variation of the substrate analog peptide moieties of the DP IV-inhibitors increases their ability to inhibit the enzyme to a remarkable extent. Possible reaction pathways are discussed.

13C and 15N NMR study of 1,2,4-Triazolo[1,5-a]pyrimidines with one tautomerism-introducing substituent

Abstract The 13 C NMR spectra of a large variety of 7-OH (7-SH, 7-NH 2 ) substituted 1,2,4-triazolo[1,5- a ]pyrimidines were studied and assigned by a whole arsenal of 2D NMR methods. The C-7 chemical shifts were not sufficiently characteristic to differentiate the lactim and the three lactam tautomers of the compounds studied. However, the 15 N NMR resonances, which were assigned by the various possible N,H coupling constants, proved that the 7-OH- and 7-SH-substituted 1,2,4-triazolo[1,5- a ]pyrimidines exist as an equilibrium of the N 4 H and the N 3 H tautomers, which is fast on the NMR time scale. PM3 quantum-chemical calculations corroborate this result obtained experimentally.

Toxin genes on pathogenicity islands: impact for microbial evolution.

Toxin-specific genes are often located on mobile genetic elements such as phages, plasmids and pathogenicity islands (PAIs). The uropathogenic E. coli strain 536 carries two alpha-hemolysin gene clusters, which are part of the pathogenicity islands I536 and II536, respectively. Using different genetic techniques, two additional PAIs were identified in the genome of the E. coli strain 536, and it is likely that further PAIs are located on the genome of this strain. Pathogenicity islands are often associated with tRNA genes. In the case of the E. coli strain 536, the PAI-associated tRNA gene leuX, which encodes a minor leucyl-tRNA, affects the expression of various virulence traits including alpha-hemolysin production. The exact mode of action of the tRNA5Leu-dependent gene expression has to be identified in the future.

Zur theorie der α-ketosäuren : Beziehungen zwischen struktur und UV-Spektren von α-ketosäuren und verwandten α-dicarbonylverbindungen☆

Zusammenfassung α-Ketosauren bilden in unpolaren Losungsmitteln ein stabiles Protonenchelat zwischen der Carboxylgruppe und der α-Carbonylgruppe aus, das den n-π-*-Ubergang der α-Carbonyl-gruppe im Vergleich zum Spektrum des unchelatisierten Molekuls stark bathochrom verschiebt und ausserdem eine Schwingungsfeinstruktur dieses Ubergangs hervortreten lasst. Auch die UV-Spektren der α-Ketosaureamide deuten auf eine intramolekulare -NH⋯OC-Wechselwirkung hin. Die Untersuchung des Alkylgruppeneinflusses auf die Lage des n-π*-Uberganges bei Verbindungen vom Typ R 1 COCOR 2 (R 1 = Alkyl; R 2 = OH, NH 2 und CH 3 ) zeigt, dass Methyl-diketo-verbindungen (CH 3 COCOR 2 ), da Hyperkonjugation moglich ist, eine Sonderstellung einnehmen.