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

Scheiner, Steve; Kleier, Daniel A.; Lipscomb, William N.

doi:10.1073/pnas.72.7.2606

Cited by 78 publications

(30 citation statements)

References 19 publications

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“…We find (2) that the buried Asp is the stronger base of this dyad, and that it is the ultimate proton acceptor of the charge relay (3). The substrate, His-57, and the rest of the enzyme are responsible for maintaining Asp-102 away from solvent in the hydrophobic environment, and His-57 acts as a proton relay between Asp-102 and other parts of the active site.…”

Section: Discussionmentioning

confidence: 99%

“…For When CH30-adds to HCONH2 to form the "tetrahedral" adduct, we find that the MBSE of the adduct is 50 kcal/mol less than that of the reactants (7). As HCONH2 is an electronically neutral species and, as such, would not be expected to have a large MBSE, we assume that MBSE CH30CHONH2-= MBSECH3O--50 kcal/mol and scale c for the adduct accordingly, i.e., [2] C CH30CHONH2 -C CH30-X (MBSECH30CHON2-/MBSECH,-) [3] The negative of the charge of a third proton placed on N to form CH30CHONH3 was taken as q°for this species (Table 1). Since the proton affinity of the species CHsOCHO(OH)-is calculated by PRDDO to be nearly identical to that of 432…”

Section: Resultsmentioning

confidence: 99%

“…As HCONH2 is an electronically neutral species and, as such, would not be expected to have a large MBSE, we assume that MBSE CH30CHONH2-= MBSECH3O--50 kcal/mol and scale c for the adduct accordingly, i.e., [2] C CH30CHONH2 -C CH30-X (MBSECH30CHON2-/MBSECH,-) [3] The negative of the charge of a third proton placed on N to form CH30CHONH3 was taken as q°for this species (Table 1). Since Table 2.…”

Section: Resultsmentioning

confidence: 99%

“…The formation of a tetrahedral intermediate in the acylation step of trypsin was investigated in Paper I (2), in which molecular orbital methods were used to study the charge relay system (3) and the attack of the serine residue on the scissile peptide linkage of the substrate. We report here a continuation of this work which includes both the acylation and deacylation steps in their entireties.…”

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confidence: 99%

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Molecular orbital studies of enzyme activity: catalytic mechanism of serine proteinases.

Scheiner¹,

Lipscomb²

1976

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

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The catalytic activity of the serine protein. ases is studied using molecular orbital methods on a model of the enzyme-substrate complex. A mechanism is employed in which Ser-195, upon donating a proton to the His-57-Asp-102 dyad, attacks the substrate to form the tetrahedral intermediate. As His-57 then donates a proton to the leaving group, the intermediate decomposes to the acyl enzyme. An analogous process takes place during deacylation, as a water molecule takes the place of Ser-195 as the nucleophile. The motility of the histidine is found to be an important factor in both steps. An attempt is made to include the effects of those atoms not explicitly included in the calculations and to comnpare the reaction rate of the proposed mechanism with that of the uncatalyzed hydrolysis. This mechanism is found to be in good agreement with structural and kinetic data.It has been determined that the chymotrypsin-catalyzed hydrolysis of substrates proceeds through an acyl enzyme intermediate (1). The formation of a tetrahedral intermediate in the acylation step of trypsin was investigated in Paper I (2), in which molecular orbital methods were used to study the charge relay system (3) and the attack of the serine residue on the scissile peptide linkage of the substrate. We report here a continuation of this work which includes both the acylation and deacylation steps in their entireties. The principal method used is partial retention of diatomic differential overlap (PRDDO), which closely reproduces minimum basis set (MBS) ab initio results (4). Also included are terms which correct the errors introduced by use of an MBS. RESULTSThe active site residues of trypsin were modeled as follows: by methanol, His-57 by imidazole, the Asp-102 anion by formate, and the scissile peptide linkage of the substrate by formamide. As described in Paper I, these molecules were then superimposed onto Huber's x-ray structure of the complex of bovine trypsin with bovine pancreatic trypsin inhibitor (5). In this structure, the scissile peptide link is distorted towards a tetrahedral carbon. However, when methanol is included at its x-ray position, a geometry optimization of formamide yielded a very nearly planar carbonyl group (see Fig. 1A Cg-H5l bond axis (corresponding to the CO-Ca axis of is that of the x-ray structure. The configuration thus obtained, analogous to the bound enzyme-substrate Michaelis complex, E-S, is shown as A in Fig. 1. We followed the formation of the tetrasubstituted intermediate (TI) as the composite of two separate processes. The first of these involved proton transfers from methanol to Nd2 of imidazole and, simultaneously, from N1' of imidazole to Oa2 of formate. The remaining geometry changes involved in the formation of the TI comprise the second. The most significant motions included here were (1) the rotation of methoxide around the C5-Hrl bond axis by 300 to attack Cf, (2) a motion of Cf up out of the carbonyl plane towards the approaching Os to form the tetrahedral adduct, and (3) the rotation o...

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Section: Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Molecular orbital studies of enzyme activity: catalytic mechanism of serine proteinases.

Scheiner¹,

Lipscomb²

1976

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

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“…The hypothesis that histidyl residues in the catalytic triads ofserine proteases are abnormally weak bases, whereas the corresponding aspartic acid residues are abnormally weak acids, has received considerable support, both experimental (13)(14)(15)(16)(17)(18) and theoretical (19)(20)(21)(22)(23). There are, however, other experimental results (24)(25)(26)(27)(28) that indicate more normal ionization behavior; at one time, substantial controversy on this point existed.…”

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confidence: 96%

Catalytic mechanism of serine proteases: reexamination of the pH dependence of the histidyl 1J13C2-H coupling constant in the catalytic triad of alpha-lytic protease.

Bachovchin¹,

Kaiser²,

Richards³

et al. 1981

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

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L-Histidine, 90% 13C enriched at the C2 position, was incorporated into the catalytic triad of a-lytic protease (EC 3.4.21.12) with the aid of a histidine-requiring mutant ofLysobacter enzymogenes (ATC 29487), and the pH dependence of the coupling constant between this carbon atom and its directly bonded proton was reinvestigated. The high degree ofspecific '3C isotopic enrichment attainable with the auxotroph permits direct observation and measurement of this coupling constant in protoncoupled '3C NMR spectra at 67.89 MHz and at 15.1 MHz. In contrast to the earlier study, the present results indicate that this coupling constant does respond to a microscopic ionization with pKa near 7.0; moreover, the magnitude of the values of 'Jc-i observed are in accord with those expected for titration of the histidyl residue. We conclude thatthe original measurement must be in error and that this coupling constant now also supports a histidyl residue that titrates more or less normally as a component of the catalytic triad of serine proteases.A "catalytic triad" comprised ofthe side-chain functional groups of aspartic acid, histidine, and serine has thus far proved to be an invariant feature of the active sites of serine proteinases as demonstrated by x-ray diffraction studies (1-6). The ubiquity and diversity ofindividual enzymes belonging to this class suggests that this array of Asp-His-Ser residues possesses special catalytic properties. The precise mode ofoperation of this triad in serine protease-catalyzed hydrolysis of amides and esters is, therefore, of considerable interest.A prerequisite to the understanding of the effectiveness of this triad is a knowledge of the ionization behavior of its component functional groups, and this has been a controversial issue. A histidyl residue is essential for activity (7-10), and because the activities of serine proteinases increase with pH in a manner indicative of the titration ofa single group having a pKa -7.0 (11), this ionization was originally assumed to represent that of the particular histidyl residue. However, Hunkapiller et at (12) proposed that this pKa of 7.0 should instead be assigned to the aspartic acid residue and that the histidyl residue should be assigned a pKa ofless than 4.0. The experimental basis for this proposal was a determination that the coupling constant between C2 ofthe histidyl residue in the catalytic triad ofa-lytic protease and its directly bonded proton was independent ofpH over the range 4.0-8.0 and indicative of a neutral imidazole ring. The result of this effective reversal of normal pKa assignments is. to make the aspartic acid carboxylate the ultimate charge donor in the operation of the so-called "charge-relay" mechanism (1, 12) of attack on the peptide bond.The hypothesis that histidyl residues in the catalytic triads ofserine proteases are abnormally weak bases, whereas the corresponding aspartic acid residues are abnormally weak acids, has received considerable support, both experimental (13-18) and theoretical (19-23). There...

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Influence of environment on proton-transfer mechanisms in model triads from theoretical calculations

Maigret

Rinaldi

et al. 1998

J. Comput. Chem.

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

Cited by 78 publications

References 19 publications

Molecular orbital studies of enzyme activity: catalytic mechanism of serine proteinases.

Molecular orbital studies of enzyme activity: catalytic mechanism of serine proteinases.

Catalytic mechanism of serine proteases: reexamination of the pH dependence of the histidyl 1J13C2-H coupling constant in the catalytic triad of alpha-lytic protease.

Influence of environment on proton-transfer mechanisms in model triads from theoretical calculations

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