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V Renner

Publications and source records attributed to V Renner.

7 recordsLinked to original sources
PubMed2001

Structures of prolyl oligopeptidase substrate/inhibitor complexes. Use of inhibitor binding for titration of the catalytic histidine residue.

Structure determination of the inactive S554A variant of prolyl oligopeptidase complexed with an octapeptide has shown that substrate binding is restricted to the P4-P2' region. In addition, it has revealed a hydrogen bond network of potential catalytic importance not detected in other serine peptidases. This involves a unique intramolecular hydrogen bond between the P1' amide and P2 carbonyl groups and another between the P2' amide and Nepsilon2 of the catalytic histidine 680 residue. It is argued that both hydrogen bonds promote proton transfer from the imidazolium ion to the leaving group. Another complex formed with the product-like inhibitor benzyloxycarbonyl-glycyl-proline, indicating that the carboxyl group of the inhibitor forms a hydrogen bond with the Nepsilon2 of His(680). Because a protonated histidine makes a stronger interaction with the carboxyl group, it offers a possibility of the determination of the real pK(a) of the catalytic histidine residue. This was found to be 6.25, lower than that of the well studied serine proteases. The new titration method gave a single pK(a) for prolyl oligopeptidase, whose reaction exhibited a complex pH dependence for k(cat)/K(m), and indicated that the observed pK(a) values are apparent. The procedure presented may be applicable for other serine peptidases.

Amino Acid Substitution↗
PubMed2000

The noncatalytic beta-propeller domain of prolyl oligopeptidase enhances the catalytic capability of the peptidase domain.

Prolyl oligopeptidase, which is involved in memory disorders, is a member of a new family of serine peptidases. In addition to the peptidase domain, the enzyme contains a beta-propeller, which excludes large peptides from the active site. The enzyme is inhibited with thiol reagents, possibly by reacting with Cys-255 located close to the substrate binding site. This assumption was tested with the Cys-255 --> Thr, Cys-255 --> Ala, and Cys-255 --> Ser variants of prolyl oligopeptidase. In contrast to the wild type enzyme, the Cys-255 --> Thr variant was not inhibited with N-ethylmaleimide, indicating that Cys-255, of the 16 free cysteine residues, exclusively accounts for the enzyme inhibition. Unlike the wild type enzyme that showed a doubly bell-shaped pH rate profile, the modified enzyme displayed a single bell-shaped pH dependence with benzyloxycarbonyl-Gly-Pro-naphthylamide. It was the high pH form of the enzyme that virtually disappeared with all three enzyme variants. A substantial reduction was also observed in k(cat)/K(m) for the aminobenzoyl-Ser-Pro-Phe(NO(2))-Ala-OH substrate. The high pK(a) (9.77) of Cys-255 determined by titration with N-ethylmaleimide excluded the possibility that ionization of the thiol group was responsible for generation of the two active enzyme forms. The impaired activity of the enzyme variants could be rationalized in terms of weaker binding, which manifests itself in high K(m) for substrates and high K(i) for inhibitors, like benzyloxycarbonyl-Gly-Pro-OH and aminobenzoyl-Ser-d-Pro-Phe(NO(2))-Ala-OH. It was concluded that, besides selecting substrates by size, the beta-propeller domain containing Cys-255 remarkably contributed to catalysis of the peptidase domain.

Amino Acid Substitution↗
PubMed2000

Substrate- and pH-dependent contribution of oxyanion binding site to the catalysis of prolyl oligopeptidase, a paradigm of the serine oligopeptidase family.

Prolyl oligopeptidase, an enzyme implicated in memory disorders, is a member of a new serine peptidase family. Crystallographic studies (Fülöp et al., 1998) revealed a novel oxyanion binding site containing a tyrosine residue, Tyr473. To study the importance of Tyr473 OH, we have produced prolyl oligopeptidase and its Tyr473Phe variant in Escherichia coli. The specificity rate constant, k(cat)/Km, for the modified enzyme decreased by a factor of 8-40 with highly specific substrates, Z-Gly-Pro-Nap, and a fluorogenic octapeptide. With these compounds, the decline in k(cat) was partly compensated for by reduction in Km, a difference from the extensively studied subtilisin. With the less specific suc-Gly-Pro-Nap, the Km value, which approximates Ks, was not significantly changed, resulting in greater diminution (approximately 500-fold) in k(cat)/Km. The second-order rate constant for the reaction with Z-Pro-prolinal, a slow tight-binding transition-state analogue inhibitor, and the Ki values for a slow substrate and two product-like inhibitors were not significantly affected by the Tyr473 OH group. The mechanism of transition-state stabilization was markedly dependent upon the nature of substrate and varied with pH as the enzyme interconverted between its two catalytically competent forms.

Amino Acid Sequence↗
PubMed1992

[Oxygen affinity and regeneration capacity of SAG-M and PAGGS-M stored erythrocytes].

In order to estimate the leftward shift of the oxygen dissociation curve of hemoglobin following storage of red blood cells (rbc) in the additive solutions SAG-M and PAGGS-M, respectively, we performed blood gas analyses after equilibrating the cells with a gas mixture containing 4% of O2 and 5% of CO2 at pH 7.4 and 37 degrees C. Additionally, we took advantage of these nearly physiological conditions to measure the recovery of the hemoglobin function in vitro. We observed a good correlation between 2,3-BPG and p50 (i.e. the oxygen tension, at which hemoglobin is half-saturated with oxygen). Within the first 3 weeks of storage, the 2,3-BPG content fell to one fifth of its original value, whereas the p50 declined from 26.6 to about 20 mm Hg. Compared to fresh cells, rbc stored for longer than 3 weeks will thus deliver 30% less oxygen to the myocardium. The rbc's ability to restore these parameters remained unchanged throughout 7 weeks of storage.

2,3-Diphosphoglycerate↗