Effect of cysteine-25 on the ionization of histidine-159 in papain as determined by proton nuclear magnetic resonance spectroscopy. Evidence for a histidine-159-cysteine-25 ion pair and its possible role in catalysis

Lewis, Sidney D.; Johnson, Frederick A.; Shafer, Jules A.

doi:10.1021/bi00504a009

Cited by 165 publications

(131 citation statements)

References 5 publications

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“…Although structurally unrelated, our results indicate that this step is mechanistically related to the proteolytic reactions of the papain͞cathepsin protein family. The active sites of these proteases contain three conserved residues, C25, H159, and N175 (45-47); before substrate binding, C25 is held in an active configuration through a thiolate-imidazolium ion interaction with H159 (48). Analogously, C184 of sortase may be activated by the imidazole ring of H120, which in all probability facilitates thiolate formation and subsequent nucleophilic attack on the carbonyl carbon at the scissile peptide bond.…”

Section: Resultsmentioning

confidence: 99%

Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus

Ilangovan

Ton‐That

Iwahara

et al. 2001

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

286

366

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Surface proteins of Gram-positive bacteria play important roles during the pathogenesis of human infections and require sortase for anchoring to the cell-wall envelope. Sortase cleaves surface proteins at the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine (T) and the amino group of cell-wall crossbridges. The NMR structure of sortase reveals a unique ␤-barrel structure, in which the active-site sulfhydryl of cysteine-184 is poised for ionization by histidine-120, presumably enabling the resultant thiolate to attack the LPXTG peptide. Calcium binding near the active site stimulates catalysis, possibly by altering the conformation of a surface loop that recognizes newly translocated polypeptides. The structure suggests a mechanistic relationship to the papain͞cathepsin proteases and should facilitate the design of new antiinfective agents.

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Section: Resultsmentioning

confidence: 99%

Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus

Ilangovan

Ton‐That

Iwahara

et al. 2001

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

286

366

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“…For the two V266E mutants, the data show that pK a1 , presumably the protonation of C163 (17), decreased by at least 0.5 pH unit when compared to that of procaspase-3(D 3 A) so that it was similar to that of wild-type caspase-3. In contrast, pK a2 , presumably the deprotonation of H121 (17), remained similar to that of the procaspase-3. These properties result in broad optimal pH ranges for both precursor and mature caspase-3(V266E) ( Figure 2B and Table 2).…”

Section: Enzyme Assaysmentioning

confidence: 85%

Mutations in the Procaspase-3 Dimer Interface Affect the Activity of the Zymogen

et al. 2003

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The interface of the procaspase-3 dimer plays a critical role in zymogen maturation. We show that replacement of valine 266, the residue at the center of the procaspase-3 dimer interface, with glutamate resulted in an increase in enzyme activity of ~60-fold, representing a pseudoactivation of the procaspase. In contrast, substitution of V266 with histidine abolished the activity of the procaspase-3 as well as that of the mature caspase. While the mutations do not affect the dimeric properties of the procaspase, we show that the V266E mutation may affect the formation of a loop bundle that is important for stabilizing the active site. In contrast, the V266H mutation affects the positioning of loop L3, the loop that forms the bulk of the substrate binding pocket. In some cases, the amino acids affected by the mutations are >20 Å from the interface. Overall, the results demonstrate that the integrity of the dimer interface is important for maintaining the proper active site conformation.Programmed cell death is dependent on the maturation of the effector caspases from latent zymogens. While the procaspases are present at relatively high concentrations in the cell, it is not clear why they are enzymatically inactive. The mature caspase is a dimer of heterodimeric units containing a large subunit (17 kDa) and a small subunit (12 kDa). In the procaspase monomer, the structural units are organized as a short pro domain (28 amino acids), the large subunit, an intersubunit linker (25 amino acids), and the small subunit. Two monomers assemble into the procaspase homodimer that consists of a contiguous 12-stranded β-sheet core with several helices surrounding the β-sheet (see Figure 1). The procaspase is cleaved at D175, in the intersubunit linker, to remove the covalent connection between the subunits. The pro domain is then removed after cleavage at D9 and then at D28. Recently determined structures of procaspase-7 (1, 2) show that the core structural unit of the procaspase is comparable to that of the mature caspase (see Figure 1). The primary differences reside in five active site loops (for a review, see ref 3). Following maturation of procaspase-7, only the position of loop L1 (residues 52-67, caspase-3 numbering) remains unaltered. The data show that the procaspase is not enzymatically active because loop L3 (residues 198-213) is unraveled and positioned away from the active site, and the catalytic C163 is rotated away from solvent so that it cannot attack the substrate. The positioning of † This work was supported by a grant from the National Institutes of Health (GM065970) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptC163 is likely due to the covalent connection between loop L2 (residues 163-175) and the intersubunit linker (residues 176-192). Following cleavage at D175, loop L3 moves more than 10 Å toward the protein core to form the substrate binding pocket. Loop L2 moves toward L4, and the intersubunit linker, now called loop L2′, flips 180° to interact with loops L2 and L...

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“…It is now well established that the formation of the Cys25 (Ϫ) …His159 (ϩ) active site ion pair is predominant at physiological pH values and is a necessary condition to cysteine proteinases exhibit catalytic competence. 25,26 The residues Cys25 and His159 possess unusual pK a values of ϳ3.3 and ϳ8.3, respectively. 23,25,27,28 In fact, the ionizations of these two catalytic residues are coupled.…”

Section: Introductionmentioning

confidence: 99%

“…The desprotonation of His159 shifts the pK a of Cys25 from ϳ3.3 to ϳ7.6, and the neutralization of the (Cys25)-S Ϫ anion decreases the pK a of His159 from ϳ8.5 to ϳ4.3. 26 The factors that contribute to the formation and stabilization of the Cys25 (Ϫ) -His159 (ϩ) ion pair in the active site of cysteine proteinases are not completely understood and have been the object of many theoretical 29 -36,90,95 and experimental studies. [37][38][39][40][41] The results described in the literature indicate that one of the most important factors contributing to the stabilization of the active site ion pair is the overall enzyme electrostatic field.…”

Section: Introductionmentioning

confidence: 99%

Electrostatic properties in the catalytic site of papain: A possible regulatory mechanism for the reactivity of the ion pair

et al. 2003

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We present an analysis of the electrostatic properties in the catalytic site of papain (EC 3.4.22.2), an archetype enzyme of the C1 cysteine proteinase family, and we investigate their possible role in the formation, stabilization and regulation of the Cys25 (؊) …His159 (؉) catalytic ion pair. The electrostatic properties were computed using a reassociation method based in multicentered multipolar expansions obtained from ab initio quantum calculations of overlapping protein fragments. Solvent effects were introduced by coupling the use of multicentered multipolar expansions to two continuum boundary element methods to solve the Poisson and the linearized Poisson-Boltzmann equations. The electrostatic profile found in the proton transfer region of papain showed that this enzyme has a well-defined electrostatic environment to favor the formation and stabilization of the catalytic ion pair. The papain catalytic site electrostatic profile can be considered as an electrostatic fingerprint of the papain family with the following characteristics: (i) the presence of a net electric field highly aligned in the (Cys25)-SG3(His159)-ND1 direction; (ii) the electrostatic profile has a saddlepoint character; (iii) it is basically a local environmental effect. Furthermore, our analysis describes a possible regulatory mechanism (the E SG3 ND1 attenuation effect) controlling the ion pair reactivity and permits to infer the Asp57 acidic residue as the most probable candidate to act as the electrostatic modulator. Proteins 2003;52:236 -253.

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Effect of cysteine-25 on the ionization of histidine-159 in papain as determined by proton nuclear magnetic resonance spectroscopy. Evidence for a histidine-159-cysteine-25 ion pair and its possible role in catalysis

Cited by 165 publications

References 5 publications

Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus

Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus

Mutations in the Procaspase-3 Dimer Interface Affect the Activity of the Zymogen

Electrostatic properties in the catalytic site of papain: A possible regulatory mechanism for the reactivity of the ion pair

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