Dissociation and unfolding of cold-active alkaline phosphatase from Atlantic cod in the presence of guanidinium chloride

被引:19
作者
Asgeirsson, B [1 ]
Hauksson, JB [1 ]
Gunnarsson, GH [1 ]
机构
[1] Univ Iceland, Inst Sci, Dept Chem, IS-107 Reykjavik, Iceland
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 2000年 / 267卷 / 21期
关键词
alkaline phosphatase; cold-adaptation; guanidine; metalloenzyme; psychrophilic;
D O I
10.1046/j.1432-1327.2000.01728.x
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
Cold-adaptation of enzymes involves improvements in catalytic efficiency. This paper describes studies on the conformational stability of a cold-active alkaline phosphatase (AP) from Atlantic cod, with the aim of understanding more clearly its structural stability in terms of subunit dissociation and unfolding of monomers. AP is a homodimeric enzyme that is only active in the dimeric state. Tryptophan fluorescence, size-exclusion chromatography and enzyme activity were used to monitor alterations in conformational state induced by guanidinium chloride or urea. In cod AP, a clear distinction could be made between dissociation of dimers into monomers and subsequent unfolding of monomers (fits a three-state model). In contrast, dimer dissociation of calf AP coincided with the monophasic unfolding curve observed by tryptophan fluorescence (fits a two-state model). The Delta G for dimer dissociation of cod AP was 8.3 kcal.mol(-1), and the monomer stabilization free energy was 2.2 kcal.mol(-1), giving a total of 12.7 kcal.mol(-1), whereas the total free energy of calf intestinal AP was 17.3 kcal.mol(-1). Thus, dimer formation provided a major contribution to the overall stability of the cod enzyme. Phosphate, the reaction product, had the effect of promoting dimer dissociation and stabilizing the monomers. Cod AP has reduced affinity for inorganic phosphate, the release of which is the rate-limiting step of the reaction mechanism. More flexible links at the interface between the dimer subunits may ease structural rearrangements that facilitate more rapid release of phosphate, and thus catalytic turnover.
引用
收藏
页码:6403 / 6412
页数:10
相关论文
共 53 条
[1]  
AKIYAMA Y, 1993, J BIOL CHEM, V268, P8146
[2]   ALKALINE-PHOSPHATASE FROM ATLANTIC COD (GADUS-MORHUA) - KINETIC AND STRUCTURAL-PROPERTIES WHICH INDICATE ADAPTATION TO LOW-TEMPERATURES [J].
ASGEIRSSON, B ;
HARTEMINK, R ;
CHLEBOWSKI, JF .
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY, 1995, 110 (02) :315-329
[3]  
Bortolato M, 1999, PROTEINS, V37, P310
[4]  
BOSSI M, 1993, J BIOL CHEM, V268, P25409
[5]  
BOWEI JV, 1989, BIOCHEMISTRY-US, V28, P7139
[6]  
BOYER CA, 1995, PROTEIN STABILITY FO, P65
[7]  
Cashikar AG, 1996, J BIOL CHEM, V271, P4741, DOI 10.1074/jbc.271.9.4741
[8]   Role of the intersubunit disulfide bond in the unfolding pathway of dimeric red kidney bean purple acid phosphatase [J].
Cashikar, AG ;
Rao, NM .
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, 1996, 1296 (01) :76-84
[9]   NEGATIVE COOPERATIVITY AND HALF OF SITES REACTIVITY - ALKALINE-PHOSPHATASES OF ESCHERICHIA-COLI WITH ZN2+, CO2+, CD2+, MN2+, AND CU2+ IN ACTIVE-SITES [J].
CHAPPELE.D ;
IWATSUBO, M ;
LAZDUNSK.M .
BIOCHEMISTRY, 1974, 13 (18) :3754-3762
[10]   INTESTINAL ALKALINE-PHOSPHATASE - CATALYTIC PROPERTIES AND HALF OF SITES REACTIVITY [J].
CHAPPELE.D ;
FOSSET, M ;
IWATSUBO, M ;
GACHE, C ;
LAZDUNSKI, M .
BIOCHEMISTRY, 1974, 13 (09) :1788-1795