Specificity of α‐chymotrypsin. Dipeptide substrates

Baumann, W.; Bizzozero, Spartaco A.; Dutler, Hans

doi:10.1016/0014-5793(70)80280-0

Cited by 57 publications

(33 citation statements)

References 10 publications

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“…This reflection for substrate preference through a change in k cat and not in K m is reminiscent of earlier work with serine proteases in which synthetic substrates designed with altered binding interactions at the P1′ and subsequent downstream binding subsites have led to nonsignificant changes in the K m for these enzymes, while displaying large variations in the k cat (15)(16)(17)(18). For MccF, the order of magnitude increase in k cat for DSA as compared to ESA can be attributed to the differential mode of stabilization of the acyl-enzyme transition state (formed after the departure of the adenine sulfonamide) in the active site.…”

Section: Discussionsupporting

confidence: 55%

Structure and function of a serine carboxypeptidase adapted for degradation of the protein synthesis antibiotic microcin C7

Agarwal

Tikhonov

Metlitskaya

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Several classes of naturally occurring antimicrobials exert their antibiotic activity by specifically targeting aminoacyl-tRNA synthetases, validating these enzymes as drug targets. The aspartyl tRNA synthetase “Trojan horse” inhibitor microcin C7 (McC7) consists of a nonhydrolyzable aspartyl-adenylate conjugated to a hexapeptide carrier that facilitates active import into bacterial cells through an oligopeptide transport system. Subsequent proteolytic processing releases the toxic compound inside the cell. Producing strains of McC7 must protect themselves against autotoxicity that may result from premature processing. The mccF gene confers resistance against endogenous and exogenous McC7 by hydrolyzing the amide bond that connects the peptide and nucleotide moieties of McC7. We present here crystal structures of MccF, in complex with various ligands. The MccF structure is similar to that of dipeptide ld -carboxypeptidase, but with an additional loop proximal to the active site that serves as the primary determinant for recognition of adenylated substrates. Wild-type MccF only hydrolyzes the naturally occurring aspartyl phosphoramidate McC7 and synthetic peptidyl sulfamoyl adenylates that contain anionic side chains. We show that substitutions of two active site MccF residues result in a specificity switch toward aromatic aminoacyl–adenylate substrates. These results suggest how MccF-like enzymes may be used to avert various toxic aminoacyl–adenylates that accumulate during antibiotic biosynthesis or in normal metabolism of the cell.

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Section: Discussionsupporting

confidence: 55%

Structure and function of a serine carboxypeptidase adapted for degradation of the protein synthesis antibiotic microcin C7

Agarwal

Tikhonov

Metlitskaya

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…No hydrolysis of I and II could be observed at an enzyme concentration of 40/aM, at which the hydrolysis of Ac-Phe-Gly-NHz, the least reactive dipeptide studied so far [8], can easily be followed. At an enzyme concentration of 80/aM a reaction was observed whose recorder trace was identical to that obtained for a blank experiment in absence of substrates and represents therefore only enzyme autolysis.…”

Section: Resultsmentioning

confidence: 92%

“…Both dipeptides I and II were tested as substrates of a-chymotrypsin at pH 7.90 and 25°C in 0.2 M sodium chloride solution as described previously for other peptide substrates [4,8]. No hydrolysis of I and II could be observed at an enzyme concentration of 40/aM, at which the hydrolysis of Ac-Phe-Gly-NHz, the least reactive dipeptide studied so far [8], can easily be followed.…”

Section: Resultsmentioning

confidence: 99%

The importance of the conformation of the tetrahedral intermediate for the α‐chymotrypsin‐catalyzed hydrolysis of peptide substrates

Bizzozero¹,

Zweifel²

1975

FEBS Letters

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“…To a stirred solution of Z-Tyr-OH (18.92 g, 60.0 mmol), H-Gly-OEt -HC1 (8.50 g, 60.8 mmol), triethylamine (6.13 g, 60.5 mmol) and N-hydroxysuccinimide (7.03 g, 61.0 mmol in dioxane-water (1: 1, v/v) 120 ml, a solution of dicyclohexylcarbodiimide (12.28 g, 60.0 mmol) in dioxane (60 ml) was added at 0 "C. The reaction mixture was stirred a t 5 "C for 15 h. The crystalline dicyclohexyl urea formed was removed by filtration and the filtrate reduced to half its volume, diluted with ethanol (1 volume) and ethyl acetate (20 volumes), extracted with 1 N HC1, 1 M NaHCO,, and water, dried over Na,SO, and evaporated to dryness. The residue was crystallized from methanol : yield, Synthesis of Ac-Gly-Tyr-Gly-NH, (11) Boc-Gly-Tyr-Gly-OEt ( X I I ) . The dipeptide ethyl ester hydrochloride obtained from X (4.73 g, 11.8 mmol) by hydrogenolysis (procedure C) was coupled with Boc-Gly-ONSu (3.13 g, 11.5 mmol) in the presence of triethylamine …”

Section: General Remarksmentioning

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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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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Specificity of α‐chymotrypsin. Dipeptide substrates

Cited by 57 publications

References 10 publications

Structure and function of a serine carboxypeptidase adapted for degradation of the protein synthesis antibiotic microcin C7

Structure and function of a serine carboxypeptidase adapted for degradation of the protein synthesis antibiotic microcin C7

The importance of the conformation of the tetrahedral intermediate for the α‐chymotrypsin‐catalyzed hydrolysis of peptide substrates

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

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