Generation of nucleophilic character in the Cys25/His159 ion pair of papain involves Trp177 but not Asp158.
Studies on papain (EC 3.4.22.2), the most thoroughly investigated member of the cysteine proteinase superfamily, have contributed substantially to our understanding of the roles of noncovalent interactions in enzyme active center chemistry. Previously, we reported evidence that the long-held view that catalytic competence develops synchronously with formation of the catalytic site (Cys25)-S-/(His159)-Im+H ion pair is incorrect and that conformational rearrangement is necessary for each of the partners to play its role in catalysis. A decrease in the level of mutual solvation of the partners of the noncatalytic "intimate" ion pair should release the nucleophilic character of (Cys25)-S- and allow association of (His159)-Im+H with the leaving group of a substrate to provide its general acid-catalyzed elimination. Hypotheses by which this could be achieved involve electrostatic modulation of the ion pair and perturbation of its hydrophobic shielding from solvent by Trp177. The potential electrostatic modulator closest to the catalytic site is Asp158, the mutation of which to Ala substantially decreases catalytic activity. Here we report an investigation of these hypotheses by a combination of computer modeling and stopped-flow pH-dependent kinetic studies using a new series of cationic aminoalkyl 2-pyridyl disulfide time-dependent inhibitors as reactivity probes. These probes 2-4 (n = 2-4), which exist as equilibrium mixtures of H3N+-[CH2]n-S-S-2-pyridyl+H and H3N+-[CH2]n-S-S-2-pyridyl which predominate in acidic and weakly alkaline media, respectively, were shown by modeling and kinetic analysis to bind with various degrees of effectiveness near Asp158 and in some cases also near Trp177. Kinetic analysis of the reactions of 2-4 and of the reaction of CH3-[CH2]2-S-S-2-pyridyl+H <==>CH3-[CH2]2-S-S-2-pyridyl 1 and normal mode calculations lead to the conclusion that Asp158 is not involved in the generation of nucleophilic character in the ion pair and demonstrates a key role for Trp177.
Gul
Sheraz
S
Laboratory of Structural and Mechanistic Enzymology, School of Biological and Chemical Sciences, Fogg Building, Queen Mary, University of London, Mile End Road, London E1 4NS, UK.
Hussain
Syeed
S
Thomas
Mark P
MP
Resmini
Marina
M
Verma
Chandra S
CS
Thomas
Emrys W
EW
Brocklehurst
Keith
K
D001224:Aspartic Acid / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010206:Papain / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D055672:Static Electricity; D014364:Tryptophan / Q000737:chemistry*
D001224:Aspartic Acid; D006639:Histidine; D014364:Tryptophan; D010206:Papain; D003545:Cysteine