LYSINE-274 IS ESSENTIAL FOR FRUCTOSE 2,6-BISPHOSPHATE INHIBITION OF FRUCTOSE-1,6-BISPHOSPHATASE

Citation
Mr. Elmaghrabi et al., LYSINE-274 IS ESSENTIAL FOR FRUCTOSE 2,6-BISPHOSPHATE INHIBITION OF FRUCTOSE-1,6-BISPHOSPHATASE, The Journal of biological chemistry, 267(10), 1992, pp. 6526-6530
Citations number
27
Language
INGLESE
art.tipo
Article
ISSN journal
0021-9258
Volume
267
Issue
10
Year of publication
1992
Pages
6526 - 6530
Database
ISI
SICI code
0021-9258(1992)267:10<6526:LIEFF2>2.0.ZU;2-E
Abstract
Lysine 274 is conserved in all known fructose-1,6-bisphosphatase seque nces. It has been implicated in substrate binding and/or catalysis on the basis of reactivity with pyridoxal phosphate as well as by x-ray c rystallographic analysis. Lys274 of rat liver fructose-1,6-bisphosphat ase was mutated to alanine by the polymerase chain reaction, and the T 7-RNA polymerase-transcribed construct containing the mutant sequence was expressed in Escherichia coli. The mutant and wild-type forms of t he enzyme were purified to homogeneity, and their specific activity, s ubstrate dependence, and inhibition by fructose 2,6-bisphosphate and A MP were compared. While the mutant exhibited no change in maximal velo city, its K(m) for fructose 1,6-bisphosphate was 20-fold higher than t hat of the wild-type, and its K(i) for fructose 2,6-bisphosphate was i ncreased 1000-fold. Consistent with the unaltered maximal velocity, th ere were no apparent difference between the secondary structure of the wild-type and mutant enzyme forms, as measured by circular dichroism and ultraviolet difference spectroscopy. The K(i) for the allosteric i nhibitor AMP was only slightly increased, indicating that Lys274 is no t directly involved in AMP inhibition. Fructose 2,6-bisphosphate poten tiated AMP inhibition of both forms, but 500-fold higher concentration s of fructose 2,6-bisphosphate were needed to reduce the K(i) for AMP for the mutant compared to the wild-type. However, potentiation of AMP inhibition of the Lys274 --> Ala mutant was evident at fructose 2,6-b isphosphate concentrations (approximately 100-mu-M) well below those t hat inhibited the enzyme, which suggests that fructose 2,6-bisphosphat e interacts either with the AMP site directly or with other residues i nvolved in the active site-AMP synergy. The results also demonstrate t hat although Lys274 is an important binding site determinant for sugar bisphosphates, it plays a more significant role in binding fructose 2 ,6-bisphosphate than fructose 1,6-bisphosphate, probably because it bi nds the 2-phospho group of the former while other residues bind the 1- phospho group of the substrate. It is concluded that the enzyme utiliz es Lys274 to discriminate between its substrate and fructose 2,6-bisph osphate.