scholarly journals Acyl and amino intermediates in reactions catalysed by pig pepsin. Analysis of transpeptidation products

1976 ◽  
Vol 153 (3) ◽  
pp. 691-699 ◽  
Author(s):  
T T Wang ◽  
T Hofmann
Keyword(s):  
Reaction Rates ◽  
Major Product ◽  
Conclusive Evidence ◽  
Acyl Transfer ◽  
High Yield ◽  
Amino Acid Residues ◽  
Small Peptides ◽  
Ph Optimum ◽  
Covalent Intermediate ◽  
Hydrolysis Of

The action of pig pepsin on a variety of small peptides including Leu-Trp-Met-Arg, Leu-Trp-Met, Leu-Leu-NH2, benzyloxycarbonyl-Phe-Leu and Gly-Leu-Tyr was studied. Leu-Leu-Leu was found to be the major product from the substrates Leu-Trp-Met-Arg and Leu-Trp-Met, indicating that the predominant reaction at pH 3.4 was a transpeptidation of the acyl-transfer type. Leu-Leu-Leu was also formed in high yield by amino transfer from benzyloxycarbonyl-Phe-Leu. Like the amino-transfer reactions the acyl transfer proceeded via a covalent intermediate, since [14C]leucine was not incorporated into transpeptidation products and did not exchange with enzyme-bound leucine in the presence of acceptors. With Leu-Trp-Met both acyl and amino transpeptidation products, namely Leu-Leu, Leu-Leu-Leu, Met-Met and Met-Met-Met, were formed in addition to methionine and leucine. With Leu-Trp-Met-Arg (1 mM) the pH optimum for the rates of hydrolysis and acyl transfer is about pH 3.4. At this pH the rate of acyl transfer exceeds that of hydrolysis; at pH 2, however, hydrolysis was faster than transfer. A comparison of the effect of the length of substrates and products on the reaction rates allows the conclusion that the binding site can extend over eight to nine amino acid residues. Although the experiments provide no conclusive evidence for or against the involvement of amino and/or acyl intermediates in the hydrolysis of long peptides and proteins, the high yield of transpeptidation reactions of both types observed with some substrates suggests a major role for the intermediates in pepsin-catalysed reactions. The results also show that when pig pepsin is used for the digestion of proteins for sequence work, the likelihood of the formation of transpeptidation products is considerable. In this way peptides not present in the original sequence could easily form in a reasonably good yield.

Acyl and amino intermediates in penicillopepsin-catalysed reactions and activation by nonsubstrate peptides

1977 ◽  
Vol 55 (4) ◽  
pp. 286-294 ◽  
Author(s):  
Tusn. T. Wang ◽  
Theo Hofmann
Keyword(s):  
Amino Acid ◽  
Proteolytic Activity ◽  
Acyl Transfer ◽  
High Yield ◽  
Major Conclusion ◽  
Small Peptides ◽  
Trypsinogen Activation ◽  
The Third ◽  
Effects Of Ph ◽  
Relative Specificity

The action of penicillopepsin on a variety of small peptides was studied qualitatively and kinetically. With the substrate benzyloxycarbonyl-Phe-Leu, benzyloxycarbonyl-Tyr-Leu and acetyl-Phe-Tyr the formation of Leu-Leu from the first two and Tyr-Tyr from the third substrate was observed. These reactions presumably proceed via a covalent amino-intermediate in analogy to pig pepsin. However, with penicillopepsin the yields of transpeptidation are low. In contrast, transpeptidations via acyl transfer proceed in high yield with a variety of substrates. With Leu-Trp-Met both acyl and amino transfer occurs as shown by the formation of Leu-Leu and Met-Met; unlike with pig pepsin, however, no Leu-Leu-Leu or Met-Met-Met was observed. Nonsubstrate peptides activate the cleavage of small substrates. (The term 'cleavage' is used here to imply that the reaction is either a hydrolysis or a transpeptidation, or both. The term 'hydrolysis' will only be used for strictly hydrolytic reactions.) As with pig pepsin the activators increase predominantly the transpeptidation reaction and have only small effects on hydrolysis. The activators increase kcat but have no effect on Km. Studies of the cleavage of six different peptides show that at pH 4.7 Km is lower than at pH 3.4 while kcat is unaffected. As with pig pepsin activation by nonsubstrate peptides is greater at pH 4.7 than at pH 3.4. Benzyloxycarbonyl-Glu-Tyr which is an inhibitor of trypsinogen activation (Ki = 50 μM) is an activator for the cleavage of Leu-Tyr-NH2 (Ka = 500 μM). Pepstatin, an inhibitor of the proteolytic activity of acid proteases, also inhibits the action of penicillopepsin on Leu-Tyr-NH2.The major conclusion from these studies is that the action of penicillopepsin on small peptides is qualitatively similar to that of pig pepsin. Transpeptidation reactions of both the amino acid and the acyl transfer type have been observed. However, there are considerable differences in the effects of pH, and in relative specificity between the two enzymes.

scholarly journals Peptidase Activity of Amidinated Thrombin

1977 ◽  
Author(s):  
E.P. Kang
Keyword(s):  
Amino Acid ◽  
Catalytic Efficiency ◽  
Enzymic Activity ◽  
Peptidase Activity ◽  
Amino Acid Residues ◽  
Small Peptides ◽  
Human Thrombin ◽  
Small Change ◽  
Hydrolysis Of ◽  
Hydrolysis Activity

Human thrombin, free of plasminogen and plasmin, was treated with ethyl acetimidate hydrochloride in order to modify the lysyl residues of the protein. By monitoring the enzymic activity in the modification mixture, it was found that the reaction was completed in about one hour and the loss of activity of thrombin was proportional to the amount of modification. After the removal of the excess ethyl acetimidate, approximately 25% of the clotting activity and of the hydrolysis activity for small peptides remained. Amino acid analysis of this modified thrombin indicated about 80% of the lysyl residues had been modified with no apparent change of other amino acid residues. By studying the thrombolytic hydrolysis of Bz-phe-val-arg-pNA, the kcat of the amidinated thrombin was about 8% of the control while the KM Secreased to 0.056 μM from 0.098 μM. The modification of the lysyl residues of thrombin, therefore, has lowered the catalytic efficiency of the enzyme with a rather small change in binding affinity. This suggests that modification of lysyl residues in the neighborhood of the active site hinders the catalytic hydrolysis of the small peptides.

scholarly journals Mode of cleavage of pig big endothelin-1 by chymotrypsin. Production and degradation of mature endothelin-1

1990 ◽  
Vol 270 (2) ◽  
pp. 541-544 ◽  
Author(s):  
M Takaoka ◽  
Y Miyata ◽  
Y Takenobu ◽  
R Ikegawa ◽  
Y Matsumura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Major Product ◽  
Proteolytic Processing ◽  
Intermediate Form ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Disulphide Bridge ◽  
Endothelin 1 ◽  
Hydrolysis Of

Pig endothelin-1 [ET-1-(1-21)] seems to be produced via proteolytic processing between Trp-21 and Val-22 of an intermediate form consisting of 39 amino acid residues, termed big ET-1-(1-39), by a chymotrypsin-like proteinase. We examined the chymotryptic-cleavage sites of big ET-1-(1-39) by reverse-phase h.p.l.c. and sequence analysis, and found that chymotrypsin cleaved initially the Tyr-31-Gly-32 bond of big ET-1-(1-39), followed by cleavage between Trp-21 and Val-22. Furthermore, chymotrypsin hydrolysed the generated ET-1-(1-21), producing a single major product that had the same amino acid sequence as ET-1-(1-21) with a cleavage between Tyr-13 and Phe-14. The disulphide bridge between Cys-1 and Cys-15 remained intact. These results indicate that the conversion of big ET-1-(1-39) into ET-1-(1-21) catalysed by chymotrypsin requires hydrolysis of the Tyr-31-Gly-32 bond before that of the Trp-21-Val-22 bond, an event followed by cleavage between Tyr-13 and Phe-14 within the loop of ET-1-(1-21). Thus a chymotrypsin-like proteinase might be involved not only in the production but also in the degradation of ET-1-(1-21) in vivo.

Barley arabinoxylan arabinofuranohydrolases: purification, characterization and determination of primary structures from cDNA clones

2001 ◽  
Vol 356 (1) ◽  
pp. 181-189 ◽  
Author(s):  
Robert C. LEE ◽  
Rachel A. BURTON ◽  
Maria HRMOVA ◽  
Geoffrey B. FINCHER
Keyword(s):  
Catalytic Efficiency ◽  
Exchange Chromatography ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Hordeum Vulgare L ◽  
Ph Optimum ◽  
Catalytic Rate Constant ◽  
Arabinoxylan Arabinofuranohydrolase ◽  
Mature Enzyme ◽  
Hydrolysis Of

A family 51 arabinoxylan arabinofuranohydrolase, designated AXAH-I, has been purified from extracts of 7-day-old barley (Hordeum vulgare L.) seedlings by fractional precipitation with (NH4)2SO4 and ion-exchange chromatography. The enzyme has an apparent molecular mass of 65kDa and releases l-arabinose from cereal cell wall arabinoxylans with a pH optimum of 4.3, a catalytic rate constant (kcat) of 6.9s−1 and a catalytic efficiency factor (kcat/Km) of 0.76 (ml·s−1·mg−1). Whereas the hydrolysis of α-l-arabinofuranosyl residues linked to C(O)3 of backbone (1 → 4)-β-xylosyl residues proceeds at the fastest rate, α-l-arabinofuranosyl residues on doubly substituted xylosyl residues are also hydrolysed, at lower rates. A near full-length cDNA encoding barley AXAH-I indicates that the mature enzyme consists of 626 amino acid residues and has a calculated pI of 4.8. A second cDNA, which is 81% identical with that encoding AXAH-I, encodes another barley AXAH, which has been designated AXAH-II. The barley AXAHs are likely to have key roles in wall metabolism in cereals and other members of the Poaceae. Thus the enzymes could participate in the modification of the fine structure of arabinoxylan during wall deposition, maturation or expansion, or in wall turnover and the hydrolysis of arabinoxylans in germinated grain.

Sulfur-Free Penicillin Derivatives. V. Preparation, Hydrolysis, and Oxidative Rearrangement Of Fused Oxazoline–Azetidinones

1975 ◽  
Vol 53 (4) ◽  
pp. 497-512 ◽  
Author(s):  
Saul Wolfe ◽  
Shui-Lung Lee ◽  
Jean-Bernard Ducep ◽  
Gérard Kannengiesser ◽  
Wha Suk Lee
Keyword(s):  
Carboxylic Acid ◽  
Sulfonic Acid ◽  
Acyl Transfer ◽  
High Yield ◽  
Methyl Groups ◽  
Acyl Substituent ◽  
Oxidative Rearrangement ◽  
Alternative Routes ◽  
Hydrolysis Of

Chlorinolysis of an anhydropenicillin affords a 2-(2′-chloro-3′S-amino, -acylamino or -phthalimido-4′-oxo)azetidinyl-3-methyl-2-butenoyl chloride, as a mixture of 2′R (cis) and 2′S (trans) epimers in which the 2′R epimer usually predominates. Hydrolysis of the acid chloride and treatment of the carboxylic acid with aqueous bicarbonate causes cyclization, in the case of a 3′-acylamino substituent, to a 2R-6-(1′-carboxy-2′-methyl-prop-1-enyl)-1-oxa-3,6-diaza-4S,5R-bicyclo[3,2,0]hept-2-ene-7-one. The mechanisms of these transformations are discussed, and alternative routes to such fused oxazoline–azetidinones are also presented.The oxazolines undergo ready hydrolysis to sulfonic acid salts of 2-(2′R-acyloxy-3′S-amino-4′-oxo)azetidinyl-3-methyl-2-butenoates upon treatment with the hydrate of a sulfonic acid in acetone solvent. Neutralization of these salts proceeds without O → N acyl transfer, because acylation yields a diacylated azetidinone in which the new acyl substituent is attached to nitrogen. Monobromination of the allylic methyl groups of these salts, followed by treatment with 2 molar-equiv. of triethylamine, leads to a deep-seated rearrangement, in high yield, to an oxazinone. The mechanism of this rearrangement and, in particular, the role of acylimines in this and related reactions, including the biosynthesis of penicillin, are discussed.

α-Glutamic acid-β-naphthylamidase from Neisseria catarrhalis

1969 ◽  
Vol 15 (11) ◽  
pp. 1293-1300 ◽  
Author(s):  
Sidney T. Cox ◽  
Francis J. Behal
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Glutamic Acid ◽  
Metal Ion ◽  
Gel Filtration ◽  
Aminopeptidase Activity ◽  
Amino Acid Residues ◽  
Ph Optimum ◽  
Hydrolysis Of ◽  
Derivatives Of

A second bacterial peptidase-like enzyme, arylamidase-II, has been isolated from cell free extracts of Neisseria catarrhalis. Arylamidase-II action is limited primarily to the hydrolysis of α-glutamic acid and α-aspartic acid derivatives of β-naphthylamine and short peptides of glutamic acid. The enzyme was purified 450-fold by gel filtration, ion exchange, and calcium phosphate chromatography. Its pH optimum and molecular weight were 7.7 and 170 000, respectively. Aside from its restricted substrate specificity, arylamidase-II has been found to be closely related in its mechanism of action, molecular weight, pH optimum, and metal ion dependence to arylamidase-I, which catalyzes the hydrolysis of neutral amino acid derivatives of β-naphthylamine. Arylamidase-II exhibits aminopeptidase activity, requiring the amino acid residues in the N-terminal and penultimate position to be of the L-configuration in order for hydrolysis to occur.

Polarographic study of hydrolysis of [8-lysine]vasopressin and its derivatives with blood serum of pregnant women

1980 ◽  
Vol 45 (4) ◽  
pp. 1099-1108 ◽  
Author(s):  
Mikuláš Chavko ◽  
Michal Bartík ◽  
Evžen Kasafírek
Keyword(s):  
Blood Serum ◽  
Pregnant Women ◽  
Degree Of Hydrolysis ◽  
Polarographic Study ◽  
Ph Optimum ◽  
Lysine Vasopressin ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Vasopressin Analogues

A polarographic study of the hydrolysis of [8-lysine]vasopressin and some hormonogens of the vasopressin series with the blood serum of women in the last week of pregnancy was studied. The dependence of hydrolysis on pH (pH optimum: 7.4-7.50, substrate concentration (Km 1.2 . 10-5M), pH stability and thermal stability were determined. The rate of hydrolysis of individual vasopressin analogues decreases in the order: [8-lysine]vasopressin > Nα-glycyl-prolyl[8-lysine]-vasopressin > Nα-leucyl-[8-lysine]vasopressin > Nα-alanyl-[8-lysine]vasopressin > Nα-phenyl alanyl-[8-lysine]vasopressin > Nα-diglycyl-[8-lysine]vasopressin > Nα-prolyl-[8-lysine]vasopressin > Nα-triglycyl-[8-lysine]vasopressin > Nα-sarcosyl-glycyl-[8-lysine]vasopressin. The degree of hydrolysis gradually increases to a multiple with the length of the pregnancy in consequence of the presence of oxytocine. However, vasopressin is also hydrolysed to a small extent with the enzymes from the blood sera of non-pregnant women. Under similar analytical conditions oxytocin was not hydrolysed with the sera of non-pregnant women and therefore oxytocin is a more suitable substrate than vasopressin for polarographic determination of serum oxytocinase.

Production, purification and characterization of thermomycolase, the extracellular serine protease of the thermophilic fungus Malbranchea pulchella var. sulfurea

1976 ◽  
Vol 22 (2) ◽  
pp. 165-176 ◽  
Author(s):  
Poh Seng Ong ◽  
G. Maurice Gaucher
Keyword(s):  
Serine Protease ◽  
Thermophilic Fungus ◽  
Amino Acid Residues ◽  
Submerged Cultures ◽  
Ph Optimum ◽  
Alkaline Serine Protease ◽  
Associated Production ◽  
Specificity Study ◽  
Malbranchea Pulchella ◽  
The Stability

The thermophilic fungus Malbranchea pulchella produces a single extracellular, alkaline, serine protease when grown at 45 °C, on 2% casein as sole carbon source. The growth-associated production of protease in submerged cultures was inhibited by addition of glucose, amino acids, or yeast extract. A simple four-step purification which yields homogeneous protease in 78% yield is described. The protease has an isoelectric point of 6.0, a pH optimum of 8.5, and is completely inhibited by serine protease inhibitors. A specificity study with small synthetic ester substrates indicated that the protease preferentially hydrolyzed bonds situated on the carboxyl side of aromatic or apolar amino acid residues which are not β-branched, positively charged or of the D configuration. Peptidase substrates and others such as N-acetyl-L-tyrosine-ethyl ester were not hydrolyzed. The protease was stable over a broad range of pH (6.5–9.5 at 30 °C, 20 h), and was particularly thermostable (t1/2 = 110 min at 73 °C, pH 7.4) in the presence of Ca2+ (10 mM). Macromolecules and Ca2+ also provide protection against the significant autolysis which occurs at pure protease concentrations greater than 0.01 mg/ml, as well as against surface denaturation which is enhanced by the presence of a silicone antifoam agent. Hence the stability of protease in submerged cultures is rationalized.

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