Self-glucosylation of glycogenin, the initiator of glycogen biosynthesis, involves an inter-subunit reaction

被引:31
作者
Lin, A [1 ]
Mu, J [1 ]
Yang, J [1 ]
Roach, PJ [1 ]
机构
[1] Indiana Univ, Sch Med, Dept Biochem & Mol Biol, Indianapolis, IN 46202 USA
关键词
D O I
10.1006/abbi.1998.1073
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
Glycogenin is a dimeric self-glucosylating protein involved in the initiation phase of glycogen biosynthesis. As an enzyme, glycogenin has the unusual property of transferring glucose residues from UDP-glucose to itself, forming an alpha-1,4-glycan of around 10 residues attached to Tyr194. Whether this self-glucosylation reaction is inter- or intramolecular has been debated. We used site-directed mutagenesis of recombinant rabbit muscle glycogenin-l to address this question. Mutation of highly conserved Lys85 to Gln generated a glycogenin mutant (K85Q) that had only 1-2% of the self-glucosylating activity of wild-type enzyme. Consistent with previous work, mutation of Tyr194 to Phe in a GST-fusion protein yielded a mutant, Y194F, that was catalytically active but incapable of self-glucosylation. The Y194F mutant was able to glucosylate the K85Q mutant. However, there was an initial lag in the self-glucosylation reaction that was abolished by preincubation of the two mutant proteins. The interaction between glycogenin subunits was relatively weak, with a dissociation constant inferred from kinetic experiments of around 2 mu M. We propose a model for the glucosylation of K85Q by Y194F in which mixing of the proteins is followed by rate-limiting formation of a species containing both subunit types. The results provide the most direct evidence to date that the self-glucosylation of glycogenin involves an inter-subunit reaction. (C) 1999 Academic Press.
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收藏
页码:163 / 170
页数:8
相关论文
共 27 条
[1]   TYROSINE-194 OF GLYCOGENIN UNDERGOES AUTOCATALYTIC GLUCOSYLATION BUT IS NOT ESSENTIAL FOR CATALYTIC FUNCTION AND ACTIVITY [J].
ALONSO, MD ;
LOMAKO, J ;
LOMAKO, WM ;
WHELAN, WJ .
FEBS LETTERS, 1994, 342 (01) :38-42
[2]   CATALYTIC ACTIVITIES OF GLYCOGENIN ADDITIONAL TO AUTOCATALYTIC SELF-GLUCOSYLATION [J].
ALONSO, MD ;
LOMAKO, J ;
LOMAKO, WM ;
WHELAN, WJ .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (25) :15315-15319
[3]   PROPERTIES OF CARBOHYDRATE-FREE RECOMBINANT GLYCOGENIN EXPRESSED IN AN ESCHERICHIA-COLI MUTANT LACKING UDP-GLUCOSE PYROPHOSPHORYLASE ACTIVITY [J].
ALONSO, MD ;
LOMAKO, J ;
LOMAKO, WM ;
WHELAN, WJ ;
PREISS, J .
FEBS LETTERS, 1994, 352 (02) :222-226
[4]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[5]   A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins [J].
Busch, C ;
Hofmann, F ;
Selzer, J ;
Munro, S ;
Jeckel, D ;
Aktories, K .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (31) :19566-19572
[6]   MECHANISM OF GLYCOGENIN SELF-GLUCOSYLATION [J].
CAO, YJ ;
STEINRAUF, LK ;
ROACH, PJ .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1995, 319 (01) :293-298
[7]  
CAO YJ, 1993, J BIOL CHEM, V268, P14687
[8]  
CHENG C, 1995, MOL CELL BIOL, V15, P6632
[9]   EUKARYOTIC PROTEINS EXPRESSED IN ESCHERICHIA-COLI - AN IMPROVED THROMBIN CLEAVAGE AND PURIFICATION PROCEDURE OF FUSION PROTEINS WITH GLUTATHIONE-S-TRANSFERASE [J].
GUAN, KL ;
DIXON, JE .
ANALYTICAL BIOCHEMISTRY, 1991, 192 (02) :262-267
[10]   Solid-phase glycopeptide synthesis of tyrosine-glycosylated glycogenin fragments as substrates for glucosylation by glycogenin [J].
Jansson, AM ;
Jensen, KJ ;
Meldal, M ;
Lomako, J ;
Lomako, WM ;
Olsen, CE ;
Bock, K .
JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 1, 1996, (10) :1001-1006