CHEMICAL RESCUE BY EXOGENOUS AMINES OF A SITE-DIRECTED MUTANT OF RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE THAT LACKS A KEY LYSYL RESIDUE

被引:37
作者
HARPEL, MR [1 ]
HARTMAN, FC [1 ]
机构
[1] OAK RIDGE NATL LAB,DIV BIOL,PROT ENGN PROGRAM,OAK RIDGE,TN 37831
关键词
D O I
10.1021/bi00184a026
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
Ligand binding to ribulose 1,5-bisphosphate carboxylase/oxygenase immobilizes the flexible loop 6 of the beta/alpha barrel domain in its closed conformation. Lys329, located at the apex of this loop, interacts electrostatically with Glu48 of the adjacent subunit and with the CO2-derived carboxylate of the carboxylated reaction intermediate [Knight et al. (1990) J. Mol. Biol. 215, 113-160]. Previous studies have implicated Lys329 in the addition of CO2 to the initial enediol(ate) intermediate: mutants at position 329 catalyze enolization of ribulose 1,5-bisphosphate and processing of isolated carboxyketone intermediate, but are severely impaired in overall carboxylation and the tight-binding of the carboxylated intermediate analogue 2-carboxyarabinitol 1,5-bisphosphate. Using the chemical rescue method of Toney and Kirsch [(1989) Science 243, 1485-1488], we show that these defects are partially overcome by exogenous amines. For example, ethylamine enhances the carboxylation rate of K329A by about 80-fold and strengthens complexation of 2-carboxyarabinitol 1,5-bisphosphate. The CO2/O-2 specificity of K329A is increased by amines, but remains lower than the wild-type value. Despite the pronounced enhancement of carboxylase activity, amines do not influence the rate at which ribulose 1,5-bisphosphate is enolized by K329A. Rescue of K329A follows an apparent Bronsted relationship with a beta of 1, implying complete protonation of amine in the rescued transition state. Rate saturation with respect to amine concentration and the different steric preferences for amines between K329A and K329C suggest that the amines bind to the enzyme in the position voided by the mutation. Two side products, one formed via beta-elimination of phosphate from enediol(ate) intermediate and the other linked preferentially to the oxygenase pathway, predominate in the absence of amines. Amines suppress these abortive products in favor of normal turnover products. Collectively, these results not only verify that Lys329 functions primarily in the gaseous substrate addition to the enediol(ate) but also demonstrate the residue's crucial role in stabilizing reaction intermediates.
引用
收藏
页码:5553 / 5561
页数:9
相关论文
共 69 条
  • [1] CRYSTAL-STRUCTURE OF THE ACTIVE-SITE OF RIBULOSE-BISPHOSPHATE CARBOXYLASE
    ANDERSSON, I
    KNIGHT, S
    SCHNEIDER, G
    LINDQVIST, Y
    LUNDQVIST, T
    BRANDEN, CI
    LORIMER, GH
    [J]. NATURE, 1989, 337 (6204) : 229 - 234
  • [2] A THEORETICAL-STUDY OF THE SINGLET-TRIPLET ENERGY-GAP DEPENDENCE UPON ROTATION AND PYRAMIDALIZATION FOR 1,2-DIHYDROXYETHYLENE - A SIMPLE-MODEL TO STUDY THE ENEDIOL MOIETY IN RUBISCO SUBSTRATE
    ANDRES, J
    SAFONT, VS
    QUERALT, J
    TAPIA, O
    [J]. JOURNAL OF PHYSICAL CHEMISTRY, 1993, 97 (30) : 7888 - 7893
  • [3] STRAINING THE DOUBLE-BOND IN 1,2-DIHYDROXYETHYLENE - A SIMPLE THEORETICAL-MODEL FOR THE ENEDIOL MOIETY IN RUBISCO SUBSTRATE AND ANALOGS
    ANDRES, J
    SAFONT, VS
    TAPIA, O
    [J]. CHEMICAL PHYSICS LETTERS, 1992, 198 (05) : 515 - 520
  • [4] Andrews TJ, 1987, BIOCH PLANTS, V10, P131
  • [5] BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
  • [6] SPECIFIC ACTIVATION OF A TYROSINE -] GLYCINE MUTANT OF DELTA-5-3-KETOSTEROID ISOMERASE BY PHENOLS
    BROOKS, B
    BENISEK, WF
    [J]. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1992, 184 (03) : 1386 - 1392
  • [7] TERTIARY STRUCTURE OF PLANT RUBISCO - DOMAINS AND THEIR CONTACTS
    CHAPMAN, MS
    SUH, SW
    CURMI, PMG
    CASCIO, D
    SMITH, WW
    EISENBERG, DS
    [J]. SCIENCE, 1988, 241 (4861) : 71 - 74
  • [8] REDUCED CO2/O2 SPECIFICITY OF RIBULOSE-BISPHOSPHATE CARBOXYLASE OXYGENASE IN A TEMPERATURE-SENSITIVE CHLOROPLAST MUTANT OF CHLAMYDOMONAS
    CHEN, Z
    CHASTAIN, CJ
    ALABED, SR
    CHOLLET, R
    SPREITZER, RJ
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1988, 85 (13) : 4696 - 4699
  • [9] COMPLEMENTING AMINO-ACID SUBSTITUTIONS WITHIN LOOP-6 OF THE ALPHA-BETA-BARREL ACTIVE-SITE INFLUENCE THE CO2/O2 SPECIFICITY OF CHLOROPLAST RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE
    CHEN, ZX
    YU, WZ
    LEE, JH
    DIAO, R
    SPREITZER, RJ
    [J]. BIOCHEMISTRY, 1991, 30 (36) : 8846 - 8850
  • [10] MUTATION OF ASPARAGINE-111 OF RUBISCO FROM RHODOSPIRILLUM-RUBRUM ALTERS THE CARBOXYLASE OXYGENASE SPECIFICITY
    CHENE, P
    DAY, AG
    FERSHT, AR
    [J]. JOURNAL OF MOLECULAR BIOLOGY, 1992, 225 (03) : 891 - 896