Hippuryl-L-phenylalanine

A chymotrypsin-like enzyme has been purified from sperm of the sea urchin, Hemicentrotus pulcherrimus, using tryptophan methyl ester (TrpOMe) linked to Sepharose 4B as an affinity column for chromatography and gel filtration. The isolated enzyme preparation is homogeneous in sodium dodecylsulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 18,500-19,000. This enzyme hydrolyses N-acetyl-L-tyrosine ethyl ester (AcTyrOEt) and N-benzoyl-L-tyrosine ethyl ester (BzTyrOEt); the optimal pH is 8.0. It does not hydrolyse N-benzoyl-L-arginine ethyl ester, N-alpha-toluenesulfonyl-L-arginine methyl ester, N-alpha-benzoyl-DL-arginine-p-nitroanilide, hippuryl-L-arginine or hippuryl-L-phenylalanine. The Michaelis constants for AcTyrOEt and BzTyrOEt are 0.05 mM and 0.0106 mM, respectively. The enzyme activity is inhibited completely by phenylmethylsulfonyl fluoride (PhMeSO2F), chymostatin and L-1-tosylamido-2-phenylethyl chloromethyl ketone (TosPheCH2Cl), and partially by soybean trypsin inhibitor and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TosLysCH2Cl). The enzyme is activated by CaCl2, MgCl2, NaCl and KCl, and loses its activity in 5 min at 67 degrees C. It digests the jelly coat and vitelline layer, not the fertilization membrane. The microvilli of unfertilized eggs elongate and decrease in number as the vitelline layer lyses. The vitelline layer lytic activity is inhibited completely by PhMeSO2F, TosPheCH2Cl, and chymostatin, and partially by soybean trypsin inhibitor, TosLysCH2Cl, and alpha 1-antitrypsin. We have confirmed by transmission electron microscopy that our chymotrypsin-like enzyme completely digests the vitelline layer. A result implying release of this enzyme from the acrosome vesicle is also reported.

N-benzoyl-L-phenylalanyl-L-phenylalanine is an excellent peptide substrate for carboxy-peptidase A; at 30 degrees C and pH 7.5, K(m) is 2.6 x 10(-5) M while k(cat) is 177 s(-1) (k(cat)/K(m) = 6.8 x 10(6) M(-1) s(-1)). Indole-3-acetic acid is a noncompetitive or mixed inhibitor towards the peptide and toward hippuryl-L-phenylalanine; plots of E/V vs [Inhibitor] are linear. N-Benzoyl-L-phenylalanine is a competitive inhibitor of peptide hydrolysis, and plots of E/V vs [Inhibitor] are again linear. One molecule of inhibitor binds per active site, and these inhibitors bind in different sites. At constant peptide substrate concentration and a series of constant concentrations of indole-3-acetic acid, plots of E/V vs the concentration of N-benzoyl-L-phenylalanine are linear and intersect behind the E/V axis and above the [Inhibitor] axis. This shows that both inhibitors can bind simultaneously and that binding of one facilitates the binding of the other (beta = 0.18). Employing the ester substrate hippuryl-DL,beta-phenyllactate, the same type of behavior is observed in the reverse sense; N-benzoyl-L-phenylalanine is a linear noncompetitive inhibitor and indole-3-acetic acid is a linear competitive inhibitor. Again the two inhibitor plot is linear and intersects above the [Inhibitor] axis (beta = 0.12). Previous X-ray crystallographic studies have indicated that indole-3-acetic acid binds in the hydrophobic pocket of the S'(1) site, while N-benzoyl-L-phenylalanine binds in the S(1)-S(2) site. The product complex for hydrolysis of N-benzoyl-L-phenylalanyl-L-phenylalanine (phenylalanine + N-benzoyl-L-phenylalanine) occupies both of these sites. However, the present work shows that the peptide substrate does not bind to the enzyme at pH 7.5 so as to be competitive with indole-3-acetic acid. The binding sites may be formed via conformational changes induced or stabilized by substrate and product binding.

The mechanism for inhibition of enzyme activity by excess zinc ions has been studied by kinetic and equilibrium dialysis methods at pH 8.2, I = 0.5 M. With carboxypeptidase A (bovine pancreas), peptide (carbobenzoxyglycyl-L-phenylalanine and hippuryl-L-phenylalanine) and ester (hippuryl-L-phenyl lactate) substrates were inhibited competitively by excess zinc ions. The Ki values for excess zinc ions with carboxypeptidase A at pH 8.2 are all similar [Ki = (5.2-2.6) X 10(-5) M]. The apparent constant for dissociation of excess zinc ions from carboxypeptidase A was also obtained by equilibrium dialysis at pH 8.2 and was 2.4 X 10(-5) M, very close to the Ki values above. With arsanilazotyrosine-248 carboxypeptidase A ([(Azo-CPD)Zn]), hippuryl-L-phenylalanine, carbobenzoxyglycyl-L-phenylalanine, and hippuryl-L-phenyl lactate were also inhibited with a competitive pattern by excess zinc ions, and the Ki values were (3.0-3.5) X 10(-5) M. The apparent constant for dissociation of excess zinc ions from arsanilazotyrosine-248 carboxypeptidase A, which was obtained from absorption changes at 510 nm, was 3.2 X 10(-5) M and is similar to the Ki values for [(Azo-CPD)Zn]. The apparent dissociation and inhibition constants, which were obtained by inhibition of enzyme activity and spectrophotometric and equilibrium dialysis methods with native carboxypeptidase A and arsanilazotyrosine-248 carboxypeptidase A, were almost the same. This agreement between the apparent dissociation and inhibition constants indicates that the zinc binding to the enzymes directly relates to the inhibition of enzyme activity by excess zinc ions. Excess zinc ions were competitive inhibitors for both peptide and ester substrates.(ABSTRACT TRUNCATED AT 250 WORDS)