Kinetic and thermodynamic study of a chemically modified highly active xylanase from Scopulariopsis sp: existence of an essential amino group.
The amino groups of purified least acidic xylanase (LAX) isomer and carboxyl groups of purified highly acidic xylanase (HAX) isomer from Scopulariopsis sp. were chemically modified, resulting in charge neutralization and reversal. Modification of the second amino group was accompanied by the complete loss of enzyme activity in both the absence and presence of xylose. Multiple alignments of family 10 and 11 xylanases revealed that there is a pair of fully conserved Lys residues only in family 10 members. Xylanase structures from family 10 members showed that one of the conserved Lys residues is found near the active-site cleft that makes an H-bond with the substrate. The LAX and HAX isoenzymes in which one amino and three to four carboxyl groups were modified were subjected to kinetic and thermodynamic characterization. There were no differences in pH optima between the native and modified HAX, but there was a broadening of pH optimum toward the alkaline range for charge-neutralized LAX and a double pH optimum for charge-reversed LAX. TheV max/K m of both modified LAX and HAX decreased relative to the native species. The thermodynamics of xylan hydrolysis showed that the decrease in the catalytic activity of modified LAX enzymes was entropically driven. When compared with native enzyme, the thermostabilities of modified LAX enzymes increased in the presence and decreased in the absence of substrate. The thermodynamics of kinetic stability for modified LAX enzymes revealed that this increase in thermolability was owing to the decrease in DeltaH# with a concomitant increase in DeltaS# compared with native LAX. The thermostabilities of all the modified HAX species decreased except that of charge-neutralized HAX, whose half-life significantly increased in 50% (v/v) aqueous dioxan. These results suggest that the altered properties of the modified enzymes were a result of the conformational changes brought about by chemical modification.
Afzal
Ahmed Jawaard
AJ
Department of Molecular Biology, Microbiology and Biochemistry, Southern Illinois University at Carbondale, Carbondale, IL 62901, USA. ajafzal@siu.edu [corrected]
Bokhari
Saleem Ahmed
SA
Siddiqui
Khawar Sohail
KS
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology*; D001709:Biotechnology / Q000379:methods*; D002621:Chemistry / Q000379:methods; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D013997:Time Factors; D043325:Xylan Endo-1,3-beta-Xylosidase / Q000737:chemistry*; D014995:Xylosidases / Q000737:chemistry*
D007527:Isoenzymes; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D008239:Lysine