Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Pivot Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Target Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Query: EC:3.4.21.1 (
chymotrypsin
)
10,938
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
In order to investigate how changes in the structures of side-chain aromatic groups of specific substrates influence binding and kinetic specificity in alpha
chymotrypsin
[
EC 3.4.21.1
]-catalyzed reactions, a number of nucleus-substituted derivatives of the specific ester substrates were prepared and steady-state kinetic studies were carried out at pH 6.5 and 7.8. Ac-Trp(NCps)-OMe was hydrolyzed more readily at low substrate concentration than Ac-Trp-OMe due to its smaller Km(app) value, suggesting that the bulky 2-nitro-4-carboxyphenylsulfenyl moiety interacts with outer residues rather than with those in the hydrophobic pocket and that this interaction increases the binding specificity. Inhibition experiments using the corresponding carboxylate and analogous inhibitors, however, showed that the carboxy group at the para position of the phenyl nucleus of the substituent sterically hinders association with the active site of
alpha-chymotrypsin
at pH 7.8 but not at pH 6.5. The kcat values of Ac-Trp(CHO)-0Me, Ac-Tyr(3-NO2)-OMe, and Ac-m-Tyr-OMe were much higher than those of the corresponding specific substrates, indicating that derivatives with a substitute as large as a formyl, nitro or hydroxyl group at the xi-position are stereochemically favorable to the catalytic process. Remarkable increases in Km(app) were also observed. The individual parameters for Ac-
Dopa
-OMe, however, were comparable to those for Ac-Tyr-OMe.
...
PMID:Kinetic study of alpha-chymotrypsin catalysis with regard to the interaction between the specificity-determining site and the aromatic side chain of substrates. 100 68
It is widely believed that hydroxyl radicals generated in vivo contribute to damage to macromolecules, such as proteins and DNA. We evaluated methodology based on the transformation of protein tyrosine to L-Dopa, via aromatic ring hydroxylation, as an index of hydroxyl radical attack on proteins. The catechol structure of
Dopa
makes it amenable to isolation with alumina, followed by HPLC analysis, typically used for the measurement of catecholamines. Because a level of controversy exists about the formation of
Dopa
by hydroxyl radicals, we conducted a systematic study of the formation of
Dopa
from tyrosine, tyrosine dipeptides, pure proteins (
chymotrypsin
and myelin basic protein), and endogenous proteins in tissue homogenates (rat brain), exposed to hydroxylating conditions (Fe2+/EDTA/ascorbate at neutral pH).
Dopa
residues in peptides and proteins were liberated by acid hydrolysis with 6 M HCl at 145 degrees C for 1 h. A marked lability of
Dopa
in 6 M HCl under hydrolysis conditions was prevented with added phenol;
chymotrypsin
and precipitated pellets of brain protein were also protective. Overall recoveries (hydrolysis plus purification procedures) averaged 83.4 +/- 1.7%. This improved analytic procedure may be useful for studying protein damage by hydroxyl radicals.
...
PMID:Protein L-dopa as an index of hydroxyl radical attack on protein tyrosine. 979 36
