Chymotrypsinogen family of proteins. IX. Steady-state kinetics of the chymotryptic hydrolysis of N-acetyl-L-tryptophan ethyl ester at pH 8.0

Rajender, Shyamala; Han, Moon Hi; Lumry, Rufus

doi:10.1021/ja00708a045

Cited by 18 publications

(15 citation statements)

References 3 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…to pucker as the tetrahedral intermediate is formed would reduce the calculated AE to zero. Our calculated energy barrier after correction for MBSE is [20][21][22][23][24][25] kcal/mole. This may be compared with the experimental enthalpy of activation of 11 kcal/mole which has been obtained for acylation of a specific ester substrate by chymotrypsin (22).…”

Section: Resultsmentioning

confidence: 99%

“…Our calculated energy barrier after correction for MBSE is [20][21][22][23][24][25] kcal/mole. This may be compared with the experimental enthalpy of activation of 11 kcal/mole which has been obtained for acylation of a specific ester substrate by chymotrypsin (22). Since the rate-determining step in acylation of esters is formation of the tetrahedral intermediate, the two results should be comparable.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Molecular orbital studies of enzyme activity: I: Charge relay system and tetrahedral intermediate in acylation of serine proteinases.

Scheiner¹,

Kleier²,

Lipscomb³

1975

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

View full text Add to dashboard Cite

The charge relay system and its role in the acylation of serine proteinases is studied using the partial retention of diatomic differential overlap (PRDDO) technique to perform approximate ab initio molecular orbital calculations on a model of the enzyme-substrate complex. The aspartate in the charge relay system is seen to act as the ultimate proton acceptor during the charging of the serine nucleophile. A projection of the potential energy surface is obtained in a subspace corresponding to this charge transfer and to the coupled motions of active site residues and the substrate. These results together with extended basis set results for cruder models suggest that a concerted transfer of protons from (1, 6). For amides, there is evidence that the rate-determining step in the formation of the acyl-enzyme complex involves the.breakdown of the tetrahedral intermediate (4, 7), and studies of substituent effects on the acylation of chymotrypsin by anilides of N-benzoyl-L-tyrosine and N-acetyl-Ltyrosine (1, 8, 9) suggest that a proton transfer is involved in the transition state of the acylation step. Recent x-ray diffraction studies of native and inhibited serine proteinases (5, 10-13) together with the kinetic studies of these enzymes and model systems (1) have led to the following proposal which elaborates the details of the acylation process. The substrate is first bound noncovalently to form a Michaelis complex, E-S. In this complex the scissile peptide linkage of the substrate is near a particularly reactive nucleophile, the hydroxyl of Ser-195. The mode of attack is not firmly resolved but one plausible proposal involves a transfer of a proton from Ser-195 either to His-57 or, through the intermediacy of His-57, to Asp 102. The Ser-195 anion would then attack the carbonyl carbon of the scissile peptide linkage to form a tetrahedral intermediate. Subsequent protonation of the leaving group by His-57 followed by cleavage of the peptide linkage then yields the acyl-enzyme. A mechanism related to the one described here for serine proteinases has recently been proposed for papain (14). In this paper we study the electronic factors responsible for the activity of the serine proteinases, using an approximate molecular orbital theory developed in this laboratory (15). This method, partial retention of diatomic differential overlap (PRDDO), has proven to be an effective method for closely reproducing minimum basis set ab initio results at relatively low cost. Here we apply the PRDDO method to the task of studying the charge relay system (10) of the serine proteinases and the energetics of the tetrahedral intermediate formation. We also use a more extended basis set to repeat certain key calculations in order to assess the probable error arising from the minimum basis set approximation.Our results may be compared with those obtained using a more approximate method (CNDO/2) (16). RESULTSIn our molecular orbital calculations, the active site residues Ser-195, His-57, and Asp-102 were modeled by methanol, imidaz...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Molecular orbital studies of enzyme activity: I: Charge relay system and tetrahedral intermediate in acylation of serine proteinases.

Scheiner¹,

Kleier²,

Lipscomb³

1975

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

View full text Add to dashboard Cite

show abstract

“…A method based on the fact that in the presence of an added nucleophile the deacylation step accelerates, was used for determining the individual kinetic constants of the enzyme reaction [8,13]. 1,4-Butanediol was used as a nucleophilic agent, because it contains two hydroxyl groups which weaken its competitive inhibiting effect on the reaction [ 14,151; to some substrates methanol was applied [13,16,17] Table I.…”

Section: Kinetic Methodsmentioning

confidence: 99%

“…We shall proceed from the fact that a-chymotryptic hydrolysis of N-acetyl-L-amino acied methyl esters involves the formation of at least three metastable intermediates (see, for example, the references in [6,8]). …”

Section: Free Energy-reaction Coordinate Projilementioning

confidence: 99%

“…To this end, we have studied the kinetics of a-chymotryptic hydrolysis of a series of N-acetyl-a-L-amino acid methyl esters, such as RCH(NHCOCH3)C(O)OCHa (Table I) and have shown how the free energy-reaction coordinate profile changes if the structure (size) of "chemically inert" hydrophobic fragment R is varied. U p till now only one free energy-reaction coordinate profile has been dealt with in the literature, that of N-acetyl-L-tryptophanate [8].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

The free energy–reaction coordinate profile for α‐chymotryptic hydrolysis of a series of N‐acetyl‐α‐L‐amino acid methyl esters

Martínek¹,

Klyosov²,

Kazanskaya³

et al. 1974

Int J of Chemical Kinetics

View full text Add to dashboard Cite

The effect of added nucleophiles (methanol and 1,4-butanediol) on the steady-state kinetics of a-chymotryptic hydrolysis of a series of -V-acetyl-L-amino acid methyl esters, R-CH(NHCOCH3)C(O)OCH3, has been studied. As a result, the rate and equilibrium consta.nts of the "elementary" steps of the enzyme process have been determined.It has also been demonstrated how the free energy-reaction coordinate profile changes if the structure (the size of the hydrocarbon chain) of the "chemically inert" substrate fragment R is varied. The effects observed can be described by the following equation:where A G~ and AGa are the free energies of formation of metastable intermediates, i.e., the enzyme-substrate complex and the acylenzyme, respectively, aGz* and aG3* are the free energies of activation for the chemical steps, i.e., enzyme acylation and acylenzyme hydrolysis, respec'ively; and AGt,,,,(R) is the free energy of transfer of substrate group R from water into a nonaqueous solvent.To explain the results obtained, a mechanism for enzyme-substrate interaction is suggested according to which the potential free energy of sorption of substrate group R on the enzyme is 2 AGtra,,(R). Such a high gain in the free energy of hydrophobic interaction may only be realized if (a) in the free enzyme the sorption region has a thermodynamically unfavorable contact with the aqueous medium, and (b) water is forced out of the active center as a result of the hydrophobic interaction of substrate group R with the enzyme.Such a model is in agreement with the published x-ray data on the structure of the crystalline enzyme.The kinetic experiment has proved that not all the potential free energy of sorption is realized as binding force. Thus the true free energy of the binding of substrate group R with tne protein does not exceed half the maximum value, both in the enzyme-substrate complex and acylenzyme.

show abstract

Protein Conformations, “Rack” Mechanisms and Water

Lumry

1971

Advances in Chemical Physics

View full text Add to dashboard Cite

Chymotrypsinogen family of proteins. IX. Steady-state kinetics of the chymotryptic hydrolysis of N-acetyl-L-tryptophan ethyl ester at pH 8.0

Cited by 18 publications

References 3 publications

Molecular orbital studies of enzyme activity: I: Charge relay system and tetrahedral intermediate in acylation of serine proteinases.

Molecular orbital studies of enzyme activity: I: Charge relay system and tetrahedral intermediate in acylation of serine proteinases.

The free energy–reaction coordinate profile for α‐chymotryptic hydrolysis of a series of N‐acetyl‐α‐L‐amino acid methyl esters

Protein Conformations, “Rack” Mechanisms and Water

Contact Info

Product

Resources

About