Isolation of acetyl peptides from acetylchymotrypsin

Oosterbaan, R.A.; Adrichem, M.E. van

doi:10.1016/0006-3002(58)90359-7

Cited by 66 publications

(13 citation statements)

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“…It has now been well established by chemical analysis that the ultimate site of specific acylation in a-chymotrypsin (Oosterbaan and Van Andrichem, 1958;Naughton et al, 1960), and a host of related enzymes, is the hydroxyl oxygen of a unique serine residue located within a specific amino acid sequence, namely, glycylaspartyl-seryl-glycine. This result, however, has been established only by chemical analysis of the denatured and degraded constituent peptides.…”

Section: Resultsmentioning

confidence: 99%

“…I corresponds very closely in its ultraviolet spectra to that of the acyl enzyme. The disparity between the spectrum of the acyl enzyme intermediate and that of the model compound, Af-acetyl-O-cinnamoylserinamide (compound II), is particularly disappointing in light of the fact that an O-acetylserine peptide has been identified (Oosterbaan and Van Andrichem, 1958) among the proteolytic degradation products of wonoacetyl chymotrypsin, an acyl enzyme isolated (Balls and Wood, 1956) under conditions similar to that employed in the spectral identification of cinnamoyl chymotrypsin. Wooten and Hess (1960) have found that even when non-ultraviolet absorbing acylating or phosphorylating agents are employed in the preparation of acyl or phosphoryl enzyme, spectral changes in the region of the tryptophan and tyrosine absorption peaks of a-chymotrypsin occur.…”

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confidence: 99%

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Spectrophotometric Identification of Acyl Enzyme Intermediates^*

Bernhard¹,

Lau²,

Noller³

1965

Biochemistry

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Spectrophotometric Identification of Acyl Enzyme Intermediates^*

Bernhard¹,

Lau²,

Noller³

1965

Biochemistry

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“…Fuller details of the buffer compositions are given in Balls, 1957), which are similar to the phosphorylated enzymes although they are much more readily reactivated. Degradation experiments have shown that in monoacetylchymotrypsin, the acetyl group is attached to the same serine hydroxyl group as is the phosphoryl group in phosphorylated chymotrypsin (Oosterbaan & van Adrichem, 1958). This evidence, together with the similar dependence on pH of phosphorylation, of acylation and of normal substrate hydrolysis (Hartley, 1956;Gutfreund & Sturtevant, 1956;Dixon & Neurath, 1957b), suggests that the initial step in each case is phosphorylation or acylation of a serine hydroxyl group eatalysed by the imidazole ring of a histidine residue.…”

Section: Discussionmentioning

confidence: 99%

The reactivation of phosphorylated chymotrypsin

Green¹,

Nicholls²

1959

Biochemical Journal

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“…It is well known that trypsin hydrolyses this quasisubstrate according to mechanism (1). The rate limiting step is deacylation and it can be slowed down by lowpH [16].It was established that p-nitrophenylacetate specifically acetylates the "active" serine in a-chymotrypsin [17,18]. Taking into account the structural analogy between trypsin and a-chymotrypsin [19], the similarity of kinetic constants for the cleavage of p-nitrophenyl acetate by both enzymes [16] we can admit, that the quasisubstrate will selectively acetylate at low p H practically the "active" serine of trypsin.…”

Section: Resultsmentioning

confidence: 99%

Catalytic Activity of Trypsin with an Acylated “Active” Serine

Bresler¹,

Krutyakov²,

Vlasov³

1971

European Journal of Biochemistry

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The applicability of the acylenzyme hypothesis to the mechanism of tryptic hydrolysis of amide and peptide bonds was studied. The "active" serine of trypsin was acylated with radicals of different size. If the acylenzyme mechanism is valid the kcat of esterase, amidase and peptidase activities of acyltrypsin must always decrease to the same extent. To decrease the degree of deacylation of carboxylic derivatives of trypsin, the enzymatic activity was measured a t 25", pH 6.0 and average incubation time 1 min. As compared with kcat of the native trypsin the values of kcat of esterase and amidase residual activities were found, respectively: for tetraformyl-trypsin 43 and 75O/,, for heptaformyl-trypsin 22 and 55OlO, for monoacetyl-trypsin 6 and 37O/,, for triacetyl-trypsin 1.4 and 22O/,, for monopropionyl-trypsin 9 and 23O/,, for mono-(dimethylpropionyl) -trypsin 12 and 12O/,, for mono-(diethylphosphory1)-or mono-( dimethylphosphory1)-trypsin 1 and lolo. Monoacetyl-trypsin demonstrated 6.401, of esterase and 5S0/, of peptidase activity. On the extrapolation to complete acylation (respectively to the zero esterase activity) the residual amidase activity decreased in the series formyl-, acetyl-, propionyl-trypsin and was equal to zero for dimethylpropionyl-trypsin. The latter acylating radical was similar in its size and hydrophobic nature to the smallest dialkylphosphoryl radicals. It is suggested that the acylenzyme mechanism is at least not a single pathway of the tryptic hydrolysis of amide and peptide bonds.Hydrolases compose a most extensively studied group of comparatively simple enzymes. The aim of this work was to elucidate the intermediate stages of amide hydrolysis catalyzed by trypsin.In the case of hydrolysis of esters, the acylenzyme mechanism is well established [ 1,2] :where E is the enzyme, S-substrate, ES-Michaelis complex, ES'-the acylated enzyme (the ester of the "active" serine in the protein with the acyl moiety of the substrate), PI is the alcohol, P2-the acid derived from the substrate.The mechanism (1) was generalized by many investigators to explain the tryptic hydrolysis of an amide bond and the related peptide bond. The strongest argument in favor of this generalization is the similar behaviour of both hydrolytic reactions of ester and amide cleavage when the "active" serine is phosphorylated by the action of diethyl-p-nitrophenylphosphate or a similar compound. The inhibition constants of both reactions are equal to the rate constant of phosphorylation of the "active" serine in the enzyme [3].This conclusion raises many objections. First of all there are no direct proofs for the applicability of .mechanism (1) for the amide and peptide hydrolysis. Then the acylating power of amides is very low. The blocking of the "active" serine in the enzyme by a bulky hydrophobic diakylphosphoryl group may interact with the hydrolysis of an amide bond by a drastic change of the tertiary structure in the vicinity of the enzyme active center (it is well known, that the conformation of diisopropylphospho...

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Isolation of acetyl peptides from acetylchymotrypsin

Cited by 66 publications

References 5 publications

Spectrophotometric Identification of Acyl Enzyme Intermediates^*

Spectrophotometric Identification of Acyl Enzyme Intermediates^*

The reactivation of phosphorylated chymotrypsin

Catalytic Activity of Trypsin with an Acylated “Active” Serine

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Isolation of acetyl peptides from acetylchymotrypsin

Cited by 66 publications

References 5 publications

Spectrophotometric Identification of Acyl Enzyme Intermediates*

Spectrophotometric Identification of Acyl Enzyme Intermediates*

The reactivation of phosphorylated chymotrypsin

Catalytic Activity of Trypsin with an Acylated “Active” Serine

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Product

Resources

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Spectrophotometric Identification of Acyl Enzyme Intermediates^*

Spectrophotometric Identification of Acyl Enzyme Intermediates^*