The Inhibition of chymotrypsins A4 and B with chloromethyl ketone reagents

1968 ◽  
Vol 46 (11) ◽  
pp. 1357-1370 ◽  
Author(s):  
Kenneth J. Stevenson ◽  
Lawrence B. Smillie
Keyword(s):  
Aromatic Ring ◽  
Histidine Residue ◽  
Straight Chain ◽  
Isolation And Identification ◽  
Chloromethyl Ketone ◽  
Basic Group ◽  
Chymotrypsin B ◽  
Chymotrypsin A

The inactivation of chymotrypsin A4 and B by the bifunctional reagents L-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone (L-TPCK) and phenoxymethyl chloromethyl ketone (PMCK) has been investigated. The rate of inactivation of chymotrypsin A4 with both reagents as a function of pH has been shown to be dependent on a basic group of pK = 6.3–6.5. Chymotrypsin B inactivation appears to be dependent on a basic group with a somewhat lower pK. For each enzyme the reaction with both reagents is associated with the loss of a single histidine residue. By the isolation and identification of 3-carboxymethylhistidine from the alkylated and oxidized peptic histidine-57 peptides, it has been concluded that both enzymes are alkylated at the nitrogen-3 position of histidine-57 by L-TPCK and PMCK. Evidence for the submolar alkylation of methionine-192 of chymotrypsin A4 by L-TPCK, PMCK, D-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone (D-TPCK), and N-methyl-L-TPCK is presented. There is no alkylation of the histidine of chymotrypsin A4 by D-TPCK or.N-methyl-L-TPCK.From a comparison of the structures of a number of reagents known to alkylate chymotrypsin A4, it has been concluded that the alkylation of methionine-192 is nonspecific and relatively independent of any defined stereochemistry of the reagent employed. To date the alkylation of histidine-57 has been shown to occur only with haloketones, and is dependent on the distance between the haloketone and the aromatic ring when the latter is present. Although the presence of an asymmetric α-carbon and acylamido group in straight-chain reagents is unnecessary for histidine alkylation, these must be of the L configuration if present.

The inhibition of chymotrypsin A4 with a homologous series of chloromethyl ketone reagents

1970 ◽  
Vol 48 (3) ◽  
pp. 364-375 ◽  
Author(s):  
Kenneth J. Stevenson ◽  
Lawrence B. Smillie
Keyword(s):  
Binding Site ◽  
Homologous Series ◽  
Imidazole Ring ◽  
The Other ◽  
Chloromethyl Ketone ◽  
Basic Group ◽  
Acid Hydrolysate ◽  
Other Hand ◽  
Hydrophobic Binding ◽  
Chymotrypsin A

The inactivation of chymotrypsin A4 (CHT-A4) by a homologous phenylalkyl series of bifunctional reagents, viz. phenyl chloromethyl ketone (PCK), benzyl chloromethyl ketone (BCK), and β-phenylethyl chloromethyl ketone (βPECK) (C6H5 [CH2]n COCH2Cl where n = 0, 1, or 2), has been investigated. The inactivation of CHT-A4 by PCK has been shown to be essentially pH independent whereas inactivation by BCK and βPECK appears to be dependent on a basic group of pK 6.0–6.3. PCK has been shown to inhibit CHT-A4 by virtue of the complete S-alkylation of methionine-192. BCK and βPECK, on the other hand, inactivate CHT-A4 through a combination of partial S-alkylation of methionine-192 (0.2 and 0.3 residue, respectively) and partial alkylation of histidine-57 (0.2 and 0.4 residue, respectively). Identification of the particular residue alkylated was obtained through the isolation and analysis of alkylated and oxidized peptic peptides obtained from the alkylated CHT-A4 by the diagonal peptide ionophoresis technique.The initial site of alkylation of BCK and βPECK on the imidazole ring of histidine-57 has not been identified. However, a unique histidine derivative has been isolated from the acid hydrolysate of oxidized histidine-57 peptide from CHT-A4–βPECK and is suggested to be 2(or 4)-hydroxymethylhistidine.On the basis of the present studies on the phenylalkyl series and on studies reported earlier on a phenylalkylamido halomethyl ketone series of bifunctional reagents which alkylates only methionine in CHT-A4, it has been suggested that alkylation of histidine by members of the phenylalkyl series is primarily a result of binding to the hydrophobic binding site of CHT-A4 rather than binding to the acylamido binding site as is suggested to be the case for the phenylalkylamido series.

scholarly journals Catalytic activity of α-chymotrypsin in which histidine-57 has been methylated

1971 ◽  
Vol 124 (1) ◽  
pp. 13-18 ◽  
Author(s):  
R. Henderson
Keyword(s):  
Catalytic Activity ◽  
Normal Function ◽  
Basic Group ◽  
Modified Enzyme ◽  
Chymotrypsin A

The properties of a derivative of α-chymotrypsin in which histidine-57 has been methylated have been examined. Although the modified enzyme binds substrate with the same affinity as does native α-chymotrypsin, acylation and deacylation occur at much decreased rates. As for native α-chymotrypsin, a basic group of pKa approx. 7 is involved in both acylation and deacylation. The significance of these results is considered in relation to the normal function of histidine-57.

Porcine Chymotrypsin A-π, a More Acidic Chymotrypsin

1975 ◽  
Vol 53 (10) ◽  
pp. 1101-1105 ◽  
Author(s):  
Eveline de Médicis ◽  
Louise Bergeron
Keyword(s):  
Steady State ◽  
Kinetic Study ◽  
Competitive Inhibitor ◽  
Catalytic Process ◽  
Binding Conformation ◽  
Bovine Trypsin ◽  
Chymotrypsin B ◽  
Chymotrypsin A ◽  
Ph Profiles

A kinetic study of porcine chymotrypsin A-π revealed two characteristic properties of this type of chymotrypsin:1. Porcine chymotrypsin A-π, like bovine chymotrypsin B-π, does not bind proflavin. which is a competitive inhibitor of bovine trypsin and chymotrypsin A-α.2. The pH profiles of the steady-state parameters show the two usual important pK's. The basic one, pK2 = 9.6, affects both Km and kcat/Km and probably controls the binding conformation of chymotrypsin. The acidic one, pK1 = 5.7, affects kcat and kcat/Km and plays a role in the catalytic process. The value of pK1 is unusually low.

Homologies in serine proteinases

1970 ◽  
Vol 257 (813) ◽  
pp. 77-87 ◽  
Keyword(s):  
Enzyme Activity ◽  
Pancreatic Juice ◽  
Serine Proteinases ◽  
Porcine Pancreas ◽  
Substrate Specificities ◽  
Elastic Protein ◽  
Chymotrypsin B ◽  
Chymotrypsin A

Bovine pancreatic juice contains approximately equal amounts of four inactive precursors of endopeptidases (zymogens): chymotrypsinogen A, chymotrypsinogen B, trypsinogen (Keller, Cohen & Neurath 1958) and a component of procarboxypeptidase which resembles a chymotrypsinogen (Brown, Greenshields, Yamasaki & Neurath 1963). In porcine pancreas another endopeptidase, elastase, is found which is uniquely effective against elastin, the elastic protein of ligaments. Chymotrypsin A and chymotrypsin B are almost identical in enzyme activity (Enenkel & Smillie 1963), but the chymotrypsins, trypsin and elastase have widely different substrate specificities, as seen, for example, in their action on the B chain of oxidized insulin (Naughton & Sanger 1961; figure 1).

Isolation and identification of chemical constituents from the roots of Peucedanum praeruptorum

Planta Medica ◽  
2015 ◽  
Vol 81 (16) ◽  
Author(s):  
YJ Lee ◽  
J Kim ◽  
J Lee ◽  
ES Cho ◽  
OS Bang
Keyword(s):  
Chemical Constituents ◽  
Isolation And Identification

In Vitro Effects of Urokinase — Prevention by Different Inhibitors

1990 ◽  
Vol 64 (03) ◽  
pp. 402-406 ◽  
Author(s):  
M D Oethinger ◽  
E Seifried
Keyword(s):  
In Vitro Study ◽  
Thrombin Time ◽  
Plasma Samples ◽  
Temperature Dependent ◽  
Chloromethyl Ketone ◽  
Control Value ◽  
Dose Time ◽  
In Vitro Effects ◽  
Full Protection

SummaryThe present in vitro study investigated dose-, time- and temperature-dependent effects of two-chain urokinase plasminogen activato(u-PA, urokinase) on normal citrated plasma. When 10 μg/ml u-PA wereadded to pooled normal plasma and incubated for 30 min at an ambient temperature (25° C), α2-antiplas-min decreased to 8% of the control value. Incubation on ice yielded a decrease to 45% of control,whereas α2-antiplasmin was fully consumed at 37° C. Fibrinogen and plasminogen fell to 46% and 39%, respectively, after a 30 min incubation at 25° C. Thrombin time prolonged to 190% of control.Various inhibitors were studied with respect to their suitability and efficacy to prevent these in vitro effects. Aprotinin exhibited a good protective effect on fibrinogen at concentrations exceeding 500 KlU/ml plasma. Its use, however, was limited due to interferences with some haemostatic assays. We could demonstrate that L-Glutamyl-L-Glycyl-L-Arginyl chloromethyl ketone (GGACK) and a specific polyclonal anti-u-PA-antibody (anti-u-PA-IgG) effectively inhibited urokinase-induced plasmin generation without interfering with haemostatic assays. The anti-u-PA-antibody afforded full protection ofα2-antiplasmin at therapeutic levels of u-PA.It is concluded that u-PA in plasma samples from patients during thrombolytic therapy may induce in vitro effects which should be prevented by the use of a suitable inhibitor such as GGACK or specific anti-u-PA-antibody.

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