Conformation and polarity of the active site of xylanase I from Thermomonospora sp. as deduced by fluorescent chemoaffinity labeling. Site and significance of a histidine residue.
A fluorescent chemoaffinity label o-phthalaldehyde (OPTA) was used to ascertain the conformational flexibility and polarity at the active site of xylanase I (Xyl I). The kinetics of inactivation of Xyl I with OPTA revealed that complete inactivation occurred due to the binding of one molecule of OPTA to the active site of Xyl I. The formation of a single fluorescent isoindole derivative corroborated these findings. OPTA has been known to form a fluorescent isoindole derivative by crosslinking the proximal thiol and amino groups of cysteine and lysine. The involvement of cysteine in the formation of a Xyl I-isoindole derivative has been negated by fluorometric and chemical modification studies on Xyl I with group-specific reagents and by amino-acid analysis. The kinetic analysis of diethylpyrocarbonate-modified Xyl I established the presence of an essential histidine at or near the catalytic site of Xyl I. Modification of histidine and lysine residues by diethylpyrocarbonate and 2,4,6-trinitrobenzenesulfonic acid, respectively, abolished the ability of the enzyme to form an isoindole derivative with OPTA, indicating that histidine and lysine participate in the formation of the isoindole complex. A mechanism for the reaction of OPTA with histidine and lysine residues present in the protein structure has been proposed. Experimental evidence presented here suggests for the first time that the active site of Xyl I is conformationally more flexible and more easily perturbed in the presence of denaturants than the molecule as a whole. The changes in the fluorescence emission maxima of a model compound (isoindole adduct) in solvents of different polarity were compared with the fluorescence behaviour of the Xyl I-isoindole derivative, leading to the conclusion that the active site is located in a microenvironment of low polarity.
George
S P
SP
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Rao
M B
MB
D000192:Actinomycetales / Q000201:enzymology*; D000345:Affinity Labels / Q000494:pharmacology*; D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry*; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007202:Indicators and Reagents / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008956:Models, Chemical; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D020539:Sequence Analysis, Protein; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013997:Time Factors; D014302:Trinitrobenzenesulfonic Acid / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry*; D009764:o-Phthalaldehyde / Q000494:pharmacology
D000345:Affinity Labels; D000596:Amino Acids; D006902:Hydroxymercuribenzoates; D007202:Indicators and Reagents; D011480:Protease Inhibitors; D013439:Sulfhydryl Reagents; C030601:4-hydroxymercuribenzoate; D006639:Histidine; D009764:o-Phthalaldehyde; D014302:Trinitrobenzenesulfonic Acid; D014508:Urea; D004228:Dithionitrobenzoic Acid; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D008239:Lysine; D003545:Cysteine; D004047:Diethyl Pyrocarbonate; D005033:Ethylmaleimide