Structural Specificity of α-Chymotrypsin: Polypeptide Substrates

Neil, Gary L.; Niemann, Carl; Hein, George E.

doi:10.1038/210903a0

Cited by 56 publications

(30 citation statements)

References 27 publications

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“…I n the case of protein [8] and peptide [9] substrates, it appears that additional residues on both sides of the specific amino acid also interact with the active site. The importance of such additional interactions, which occur in the secondary interaction range, may be investigated by extending the kinetic studies to suitable peptide substrates.…”

mentioning

confidence: 99%

“…These interactions, which occur within what can be termed the primary interaction range, have therefore been investigated kinetically in detail using derivatives of single amino acids, mainly N-acylated esters, as model substrates I n the case of protein [8] and peptide [9] substrates, it appears that additional residues on both sides of the specific amino acid also interact with the active site. The importance of such additional interactions, which occur in the secondary interaction range, may be investigated by extending the kinetic studies to suitable peptide substrates.…”

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confidence: 99%

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Kinetic Investigation of the α‐Chymotrypsin‐Catalyzed Hydrolysis of Peptide Substrates

Bizzozero¹,

Baumann²,

Dutler³

1973

European Journal of Biochemistry

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A number of peptide substrates of the general structure Ac-Lxn-. . . -Lxz-Lxl-Gly-NH, have been synthesized and their a-chymotrypsin-catalyzed hydrolyses studied. The acylation rate constants, E23 (= kcat), and the dissociation constants of the enzyme-substrate complexes, K J~A (= Km), have been determined using a modified pH-stat and a numerical method for the acquisition and processing of the data. On the basis of these constants a quantitative relationship between the peptide structure N-terminal to the cleaved bond and reactivity has been determined. The results are shown to be consistent with the enzyme-substrate interaction scheme proposed in 1971 by Segal et al. (Biochemistry 10, 3728). A comparison of the k , , l K g~ values indicates that the influence of a single structural change on the overall reactivity i s virtually independent of the nature of the remainder of the substrate. In addition a comparison of the k,, and K E A values shows that, in general, changes in substrate structure are mainly reflected by changes in k23 rather than in KEA. A few exceptions have been found: K E A or K E A and k,, change when glycine is introduced at the 22 position, when this glycine is replaced by alanine or when alanine is introduced at the 23 position.The observation that a-chymotrypsin has a marked side-chain specificity [l], in that it preferentially catalyzes the hydrolysis of peptide bonds C-terminal to aromatic amino-acid residues, led to the conclusion that the interactions between a single specific amino-acid residue and the active site of the enzyme are of primary importance in determining which peptide bond is to be cleaved. These interactions, which occur within what can be termed the primary interaction range, have therefore been investigated kinetically in detail using derivatives of single amino acids, mainly N-acylated esters, as model substrates I n the case of protein [8] and peptide [9] substrates, it appears that additional residues on both sides of the specific amino acid also interact with the active site. The importance of such additional interactions, which occur in the secondary interaction range, may be investigated by extending the kinetic studies to suitable peptide substrates. I n order to obtain kinetic data with peptides which is as accurate as that for ester substrates, it is necessary that only one peptide bond is hydrolyzed. For this purpose substrates of the general structure have been synthesized, where only Lxl is a specific amino acid residue. Thus it is expected that only the peptide bond between LZ1 and L,1 (indicated by the arrow) is cleaved during the measurements. In designing these substrates, it is important to select end groups Ex and E, which do not give rise to disturbing interactions with the enzyme.The pH-stat titration offers a simple method of analysis for ester hydrolysis. However, this method is technically more difficult to apply to peptide hydrolysis [lo], since the formation of amine product Em.

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mentioning

confidence: 99%

mentioning

confidence: 99%

Kinetic Investigation of the α‐Chymotrypsin‐Catalyzed Hydrolysis of Peptide Substrates

Bizzozero¹,

Baumann²,

Dutler³

1973

European Journal of Biochemistry

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“…This had already been demonstrated by NEtL et al (130) who showed that Val, Ile, Lys, and Pro in P2 favoured hydrolysis. The X-ray data showed that good van der Waars contacts could be obtained between a Leu in P2 and the enzyme.…”

Section: Giy216 Trp215 Ser214mentioning

confidence: 51%

“…The close resemblance is indeed impressive, but since the subsites in the two enzymes include many other side-chains the differences in specificity of Sz and S 4 (see below) is understandable since no homology exist between the mammalian and bacterial enzymes. Furthermore, nothing has been deduced from the crystallographic studies about the subsites S' and S~ in chymotrypsin observed by NEIL et al (130) and MORIHARA et aL (123). It would be interesting to know if the similarities found with S1, $2, and $3 also extend to S' and S~.…”

Section: Giy216 Trp215 Ser214mentioning

confidence: 99%

“…Recognizing that in large substrates multiple interactions between substrate and protein took place, NEIL et al (130) distinguished between primary specificity which was the ,,minimum structure required for enzymatic specificity to be manifest,, and secondary specificity which is determined by the influence of all other interactions between enzyme and substrate on the hydrolytic action. In the case of the highly specific trypsin, the term ,,specificity site, seems appropriate, but since most other enzymes, including the subtilisins, hydrolyze a variety of bonds, all the interactions between enzyme and substrate must be taken into consideration in evaluating the specificity.…”

Section: I'nh-ch-co]nh-ch-cotnh-ch-co]nh-ch-coimentioning

confidence: 99%

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Chemical modifications of the subtilisins with special reference to the binding of large substrates. A review

Svendsen

1976

Carlsberg Res. Commun.

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Subtilisin type Carlsberg and subtilisin BPN' are two well characterized extracellular proteases from Bacillus subtilis and Bacillus amyloquefaciens, respectively. The present review summarizes the various chemical modification studies which have been performed with these enzymes. Of special interest has been those modifications which lead to changes in the enzymatic properties with regard to the hydrolysis of polypeptide substrates but not small synthetic ester substrates. In this way it is possible to obtain information about how large an area of the surface of the enzyme is involved in the binding of natural substrates (e.g. clupein, gelatine, casein). Nitration, iodination, glutarylation or succinylation of the subtilisins leads to increases in the hydrolyses of clupein and gelatine, while modification of carboxyl groups leads to a decrease. In all these cases the hydrolysis of small ester substrates is almost unaffected. A section of the review deals with the comparison between the results obtained by chemical modification studies and the two other methods available for the study of "secondary binding sites": X-ray diffraction analyses and kinetic studies with synthetic polypeptides. The productive binding mode for polypeptides proposed by KRAUT and coworkers is in agreement with the results obtained by nitration of the subtilisins. However, results from our laboratory and the literature point towards more than one mode of productive binding. These results are discussed. Finally a comparison is made between the secondary binding sites in subtilisin and other proteolytic enzymes, especially the serine proteases. The importance of secondary interactions between enzyme and substrate is discussed.

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Proteinase‐Catalyzed Synthesis of Peptide Bonds

Fruton

1982

Advances in Enzymology - And Related Areas of Molecular Biology

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Structural Specificity of α-Chymotrypsin: Polypeptide Substrates

Cited by 56 publications

References 27 publications

Kinetic Investigation of the α‐Chymotrypsin‐Catalyzed Hydrolysis of Peptide Substrates

Kinetic Investigation of the α‐Chymotrypsin‐Catalyzed Hydrolysis of Peptide Substrates

Chemical modifications of the subtilisins with special reference to the binding of large substrates. A review

Proteinase‐Catalyzed Synthesis of Peptide Bonds

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