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

Bizzozero, Spartaco A.; Zweifel, Bernhard O.

doi:10.1016/0014-5793(75)80351-6

Cited by 47 publications

(18 citation statements)

References 20 publications

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“…The course of hydrolysis was followed by registering at time intervals of 5 -10 s the amount of NaOH added to maintain the pH at a constant value. Details of the registration method have already been published [21]. From the data thus collected the amounts of peptide hydrolyzed were calculated taking into account the ionisation of the amine product set free during hydrolysis according to Eqn (1)…”

Section: Kinetic Measurementsmentioning

confidence: 99%

“…Amount of Amount of pKa substrate base subjected to consumed hydrolysis Ac-Tyr-Gly -Gly-NHp Ac-Tyr-Ah-Gly-NH2 Ac-Tyr-Gly-Ala-NH2 Ac-Tyr-Ala-Ala-NHr Ac-Tyr-Gly-Gly-Gly-NH2 Ac-Tyr-Gly -Gly-Ala-NH, Ac-Tyr-Ala-Ala-Ala-NH2 Ac-Tyr-Ala-Pro-NH2 Ac-Phe-Val-NH2 and their standard deviations were determined by iterative regression [21] on Eqn (2) using first estimates of the constants obtained by linear regression on the reciprocal form (00 vs uo/[A]) of Eqn (2). The computer program also produced Eadie plots of the initial rates which served to check preliminarily the conformity of peptide hydrolysis to Michaelis-Menten kinetics.…”

Section: Substratementioning

confidence: 99%

“…Initial rates uo were computed by fitting polynomiais to the registered data according to a method previously described [21]. The kinetic constants k,,, and K,,, of the Michaelis-Menten equation Table 2.…”

Section: Kinetic Measurementsmentioning

confidence: 99%

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

Bizzozero¹,

Baumann²,

Dutler³

1982

European Journal of Biochemistry

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Peptide substrates of the general structure Ac-Tyr-Lyl-Lyz-. . -Ly,-NH2 and Ac-Phe-L,,-NHz have been synthesized and subjected to cr-chymotrypsin-catalyzed hydrolysis to collect information on the interactions between the enzyme active site and the amino-acid residues Lyl, Ly2, etc., C-terminal to the susceptible bond of the peptide. For this purpose changes in the dissociation constants of the enzyme-substrate complexes and in the rate constants of acylation have been related to the structural variations of the substrates. The results indicate that interactions occur with the two residues next to the scissible bond, L,1 and Lyz, but not with residue Ly3. Structural description of individual interactions was carried out with the aid of skeletal models of the active site. From such combination of kinetic and structural data a plausible interaction scheme for the substrate side C-terminal to the scissible bond has been deduced. This interaction scheme, which defines conformation and orientation of this part of the substrate within the active site, is characterized by the presence of a single hydrogen bond occurring between NH(Ly2) and . No donor interacting with the back-bone carbonyl groups of residues Lyl and LY2 could be detected in the model of a-chymotrypsin. The effect of modification of the side chains of residues Lyl and LY2 on the kinetic constants was shown to be consistent with the interactions assumed to occur between the side chains of these residues and the active site. The interpretation of the results obtained from these specificity studies have led to refined concepts concerning the relative importance of different sets of enzyme-substrate interactions in determining reactivity.Endopeptidases possess extended active sites which can accommodate between five and seven amino-acid residues of peptide substrates 11-51, Since they usually display a marked specificity for the side chain of the residue containing the reactive carbonyl group, the interactions of this residue with the enzyme are often termed primary interactions. For some serine proteases, as for trypsin, chymotrypsin and elastase, the structural basis of these interactions is clearly discernible from the known three-dimensional structures of the active sites. The other interacting residues of the substrate are said to form secondary interactions with the enzyme. On account of differences in the way these secondary interactions affect the rate of hydrolysis, they can be subdivided into two sets: those originating from the invariant part and those originating from the variable part of the peptide substrate. The invariant part includes the atoms of the backbone and the p carbons of the side chains, which are present in all amino acids but glycine. It is reasonable to assume that this invariant part, together with the specific residue, leads to interactions occurring according to a well defined interaction scheme which henceforth will be called the 'cardinal' interaction scheme. For chymotrypsin, such an interaction scheme regarding the substr...

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Section: Kinetic Measurementsmentioning

confidence: 99%

Section: Substratementioning

confidence: 99%

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

Bizzozero¹,

Baumann²,

Dutler³

1982

European Journal of Biochemistry

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“…Bizzozero and Dutler (1981) extended this study to suggest that a stereochemical inversion is the committing step in proteolysis; a methylated peptidebond N atom is thereby restrained from rapid inversion. The slow turnover of the reaction described here supports these hypotheses and provides a means to view structural interactions within a functioning enzyme.…”

mentioning

confidence: 95%

“…On the basis of molecular orbital considerations proposed by Deslongchamps (1975), Bizzozero and Zweifel (1975) showed that a-chymotrypsin does not cleave a peptide (or ester) bond when the "leaving-group" N (or C) atom is alkylated (Pro or sarcosine). Bizzozero and Dutler (1981) extended this study to suggest that a stereochemical inversion is the committing step in proteolysis; a methylated peptidebond N atom is thereby restrained from rapid inversion.…”

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confidence: 99%

Analysis of an enzyme-substrate complex by x-ray crystallography and transferred nuclear overhauser enhancement measurements: porcine pancreatic elastase and a hexapeptide

Meyer¹,

Clore²,

Gronenborn³

et al. 1988

Biochemistry

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The hexapeptide substrate Thr-Pro-nVal-NMeLeu-Tyr-Thr reacts with porcine pancreatic elastase sufficiently slowly that accelerated crystallographic data collection procedures and two-dimensional transferred nuclear Overhauser enhancement measurements could be used to study the geometry of binding. Both studies report a time-averaged population of the Michaelis complex state, prior to proteolysis. This result provides an important data point along the reaction coordinate pathway for serine proteases. Crystallographic data to 1.80-A resolution were used in the structure analysis with refinement to an R-factor of 0.19.W ith the exception of inhibited complexes, most enzymatic reactions are opaque to investigation of structure-functional relationships in and around the active site. A number of details about the reaction mechanism of the serine proteases therefore remain poorly understood. In this paper we describe two complementary experiments designed to probe structural details of a kinetically retarded reaction involving porcine pancreatic elastase (PPE).lOn the basis of molecular orbital considerations proposed by Deslongchamps (1975), Bizzozero and Zweifel (1975) showed that a-chymotrypsin does not cleave a peptide (or ester) bond when the "leaving-group" N (or C) atom is alkylated (Pro or sarcosine). Bizzozero and Dutler (1981) extended this study to suggest that a stereochemical inversion is the committing step in proteolysis; a methylated peptidebond N atom is thereby restrained from rapid inversion. The slow turnover of the reaction described here supports these hypotheses and provides a means to view structural interactions within a functioning enzyme. Our first attempt to test this hypothesis with PPE using as a substrate Pro-Ala-Pro-Tyr led to backward, nonproductive binding (Clore et al., 1986a). We therefore chose to study a hexapeptide, Thr-Pro-nVal-NMeLeu-Tyr-Thr, designed to bind unambiguously to porcine pancreatic elastase. As before (Clore et al., 1986a), a dual-pronged attack, using both high-resolution crystallography and N M R spectroscopy involving the use of TRNOE measurements (Clore & Gronenborn, 1982, 1983, was initiated. Because of the lability of the complex, the crystallographic data collection was limited by a regimen chosen to ensure adequate ligand binding: 24 h of soaking and 48 h of data collection. Such a regimen places a severe strain on conventional crystallographic measurements, which traditionally can require weeks to months to complete. Similarly, the N M R measurements were limited to 24 h of data collection per sample. EXPERIMENTAL PROCEDURESSamples. PPE was purchased from Serva (20931) and used without further purification. The hexapeptide Thr-PronVal-NMeLeu-Tyr-Thr was synthesized by Bachem (Switz- 0006-2960/88/0427-0725$01 .50/0 erland) and was >99.5% pure as judged by HPLC. Crystallography. Crystals of PPE were grown from a 0.1 M phosphate buffer (pH 5.0) (Meyer et al., 1986) and harvested with a 0.1 M sulfate plus 0.1 M phosphate (pH 5.0) buffer. A crystal (0...

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Refined crystal structure (2.3 �) of a double-headed winged bean ?-chymotrypsin inhibitor and location of its second reactive site

et al. 1999

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The crystal structure of a doubleheaded ␣-chymotrypsin inhibitor, WCI, from winged bean seeds has now been refined at 2.3 Å resolution to an R-factor of 18.7% for 9,897 reflections. The crystals belong to the hexagonal space group P6 1 22 with cell parameters a ‫؍‬ b ‫؍‬ 61.8 Å and c ‫؍‬ 212.8 Å .The final model has a good stereochemistry and a root mean square deviation of 0.011 Å and 1.14°from ideality for bond length and bond angles, respectively. A total of 109 ordered solvent molecules were localized in the structure. This improved structure at 2.3 Å led to an understanding of the mechanism of inhibition of the protein against ␣-chymotrypsin. An analysis of this higher resolution structure also helped us to predict the location of the second reactive site of the protein, about which no previous biochemical information was available. The inhibitor structure is spherical and has twelve antiparallel ␤-strands with connecting loops arranged in a characteristic ␤-trefoil fold common to other homologous serine protease inhibitors in the Kunitz (STI) family as well as to some non homologous functionally unrelated proteins. A wide variation in the surface loop regions is seen in the latter ones. Proteins 1999;35:321-331.1999 Wiley-Liss, Inc.

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

Cited by 47 publications

References 20 publications

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

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

Analysis of an enzyme-substrate complex by x-ray crystallography and transferred nuclear overhauser enhancement measurements: porcine pancreatic elastase and a hexapeptide

Refined crystal structure (2.3 �) of a double-headed winged bean ?-chymotrypsin inhibitor and location of its second reactive site

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