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Title 

Purification and characterization of a thermostable ADP-glucose pyrophosphorylase from Thermus caldophilus GK-24

Authors 

Jeong Heon KoChul Ho KimDae Sil LeeYu Sam Kim

Publisher 

Portland Press

Issue Date 

1996

Citation 

Biochemical Journal, vol. 319, no. 0, pp. 977-983

Keywords 

adenosine diphosphateadenosine phosphateadenosine triphosphatealanineenzyme activatorfructose 1,6 bisphosphatefructose 6 phosphateglucose 1 phosphateglucose 1 phosphate adenylyltransferaseglucose 6 phosphate

Abstract 

An extremely thermostable ADP-glucose pyrophosphorylase (AGPase) has been purified from Thermus caldophilus GK-24 to homogeneity by chromatographic methods, including gel filtration and ion-exchange and affinity chromatography. The specific activity of the enzyme was enriched 134.8-fold with a recovery of 10.5%. The purified enzyme was a single band by SDS/PAGE with a molecular mass of 52 kDa. The homotetrameric structure of the native enzyme was determined by gel filtration analysis, which showed a molecular mass of 230 kDa on a Superose-12 column, indicating that the structure of the enzyme is different from the heterotetrameric structures of higher-plant AGPases. The enzyme was most active at pH 6.0. The activity was maximal at 73-78°C and its half-life was 30 min at 95°C. Kinetic and regulatory properties were characterized. It was found that AGPase activity could be stimulated by a number of glycolytic intermediates. Fructose 6-phosphate, fructose 1,6-bisphosphate, phenylglyoxal and glucose 6-phosphate were effective activators, of which fructose 1,6-bisphosphate was the most effective. The enzyme was inhibited by phosphate, AMP or ADP. ATP and glucose 1-phosphate gave hyperbolic-shaped rate-concentration curves in the presence or absence of activator. A remarkable aspect of the amino acid composition was the existence of the hydrophobic and Ala + Gly residues. The N-terminal and internal peptide sequences were determined and compared with known sequences of various sources. It was apparently similar to those of AGPases from other bacterial and plant sources, suggesting that the enzymes are structurally related.

ISSN 

0264-6021

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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