CHARACTERIZATION AND SEQUENCING OF AN ACTIVE-SITE CYSTEINE-CONTAINING PEPTIDE FROM THE XYLANASE OF A THERMOTOLERANT STREPTOMYCES

被引:13
作者
KESKAR, SS [1 ]
RAO, MB [1 ]
DESHPANDE, VV [1 ]
机构
[1] NATL CHEM LAB,DIV BIOCHEM SCI,POONA 411008,MAHARASHTRA,INDIA
关键词
D O I
10.1042/bj2810601
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The kinetics of chemical modification of the xylanase from a thermotolerant Streptomyces T7 indicated the involvement of 1 mol of cystein residue/mol of enzyme [Keskar, Srinivasan & Deshpande (1989) Biochem. J. 261, 49-55]. The chromophoric reagent N-(2,4-dinitroanilino)maleimide (DAM) reacts covalently with thiol groups of xylanase with complete inactivation. Protection against inactivation was provide by the substrate (xylan). The purified xylanase that had been modified with DAM was digested with pepsin and the peptides were purified by gel filtration followed by peptide mapping. The active-site peptide was distinguished from the other thiol-containing peptides by comparison of the peptides generated by labelling the enzyme in the presence and in the absence of the substrate. The peptide mapping of the modified enzyme in the absence of xylan showed three yellow peptides, whereas in the presence of xylan only two yellow peptides were detected. The active-site peptide protected by the substrate failed to form the complex with DAM. The modified active-site peptide was isolated and sequenced. Gas-phase sequencing provided the following sequence: Ser-Val-Ile-Met-Xaa-Ile-Asp-His-Ile-Arg-Phe. This is the first report on the isolation and sequencing of the active-site peptide from a xylanase. The comparison of reactive cysteine-containing peptide sequence with the catalytic regions of other glucanases revealed the presence of a conserved aspartic acid residue.
引用
收藏
页码:601 / 605
页数:5
相关论文
共 30 条
[1]   PRODUCTION AND PROPERTIES OF XYLANASES FROM ACTINOMYCETES [J].
BALL, AS ;
MCCARTHY, AJ .
JOURNAL OF APPLIED BACTERIOLOGY, 1989, 66 (05) :439-444
[2]  
BASTAWDE KB, 1991, ACS SYM SER, V460, P417
[3]   MICROBIAL XYLANOLYTIC SYSTEMS [J].
BIELY, P .
TRENDS IN BIOTECHNOLOGY, 1985, 3 (11) :286-290
[4]   SUBSTRATE-BINDING SITE OF ENDO-1,4-BETA-XYLANASE OF THE YEAST CRYPTOCOCCUS-ALBIDUS [J].
BIELY, P ;
KRATKY, Z ;
VRSANSKA, M .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1981, 119 (03) :559-564
[5]   ESSENTIAL CARBOXY GROUPS IN XYLANASE-A [J].
BRAY, MR ;
CLARKE, AJ .
BIOCHEMICAL JOURNAL, 1990, 270 (01) :91-96
[6]   DETERMINATION OF DISULPHIDE GROUPS IN PROTEINS [J].
CAVALLINI, D ;
GRAZIANI, MT ;
DUPRE, S .
NATURE, 1966, 212 (5059) :294-+
[8]  
CLARKWALKER GF, 1961, J CHEM SOC, V547, P2810
[9]   CHEMICAL MODIFICATION OF XYLANASES - EVIDENCE FOR ESSENTIAL TRYPTOPHAN AND CYSTEINE RESIDUES AT THE ACTIVE-SITE [J].
DESHPANDE, V ;
HINGE, J ;
RAO, M .
BIOCHIMICA ET BIOPHYSICA ACTA, 1990, 1041 (02) :172-177
[10]  
Goodwin T. W., 1946, BIOCHEM JOUR, V40, P628