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S Kupfer

Publications and source records attributed to S Kupfer.

6 recordsLinked to original sources
PubMed1978

Kinetics of the interaction of alpha-chymotrypsin with trypsin kallikrein inhibitor (Kunitz) in which the reactive-site peptide bond Lys-15--Ala-16 is split.

Modified trypsin kallikrein inhibitor (I*), with the reactive-site peptide bond Lys-15--Ala-16 split, reacts with alpha-chymotrypsin (E) via an intermediate X to the stable tetrahedral complex C:E + I in equilibrium X leads to C. Formation X constitutes a fast pre-equilibrium (equilibrium constant Kx = 7 X 10(-5) M, association rate constant kx = 4 X 10(3)M-1s-1) to the slow reaction X leads to C (rate constant kc = 2 X 10(-3) s-1), all values at pH 7.5. No intermediate X is observed when alpha-chymotrypsin reacts with I*-OMe in which the carboxyl group of Lys-15 is esterified by methanol. This observation as well as the different pH dependence of the overall association rate constants in the case of I* and I*-OMe indicate tha formation of X precedes formation of the acyl enzyme in the catalytic pathway. The data are compared to the similar results obtained with beta-trypsin and I* or I*-OMe.

Alanine↗
PubMed1976

Hydrolysis-resynthesis equilibrium of the lysine-15--alanine-16 peptide bond in bovine trypsin inhibitor (Kunitz).

Catalytic amounts of bovine beta-trypsin, bovine alpha-chymotrypsin and porcine plasmin establish a true thermodynamic equilibrium between virgin (I) (reactive site Lys15-Ala16 peptide bond intact) and modified (I) (this bond hydrolyzed) bovine trypsin/kallikrein inhibitor (Kunitz). The very slow reaction rates for attaining equilibrium are pH-dependent and differ for different enzymes. Optimal rates are for beta-trypsin at pH 3.75, for alpha-chymotrypsin at pH 5.5, and for plasmin at pH 5.0. Under conditions of optimum pH the equilibrium is reached with the highest rate by plasmin. In 10(-5)M inhibitor solutions the equilibrium concentrations of virgin and modified inhibitor are established by plasmin after almost 300 days starting from either pure virgin or pure modified inhibitor. Thus, the hydrolysis constant KHyd = [I]/[I] is determined to be 0.33 at pH 5.0. In spite of many unsuccessful attempts, this demonstrates that the reactive site peptide bond Lys15-Ala16 in the bovine trypsin inhibitor (Kunitz) can be hydrolyzed by catalytic amounts of endopeptidase. It further confirms that the hydrolyzed Lys15-Ala16 peptide bond in modified inhibitor is subject to thermodynamic control resynthesis.

Alanine↗
PubMed1975

The effect of cleaving the reactive-site peptide bond Lys-15--Ala-16 on the conformation of bovine trypsin-kallikrein inhibitor (K unitz) as revealed by solvent-perturbation spectra, circular dichroism and fluorescence.

Spectroscopic measurements of virgin bovine trypsin-kallikrein inhibitor and its modified species (in which the reactive-site peptide bond Lys-15--Ala-16 is split) indicate a conformational difference between both proteins. The inhibitor contains four tyrosines but no tryptophan. In the modified inhibitor a tyrosyl blue shift is seen in the difference absorption spectrum of modified against virgin inhibitor. The solvent perturbation spectra show an increase of the fraction of exposed tyrosyls from 0.45 in the virgin inhibitor to 0.59 in the modified form. Comparison of the circular dichroism spectra of the modified and virgin inhibitors reveals a decrease of the mean residue ellipticity in the tyrosine and peptide bond region of the modified inhibitor. In the fluorescence spectra a 50% increase in the quantum yield of the tyrosine fluorescence is observed in the modified inhibitor. All these spectroscopic data support the idea, which is also evidenced by the X-ray crystallographic model, that in the modified inhibitor up to five residues from Ala-16 to Arg-20 gain rotational freedom.

Alanine↗