Journal article
Catalytic implications of the higher plant ADP-glucose pyrophosphorylase large subunit
Phytochemistry (Oxford), Vol.68(4), pp.464-477
2007
Handle:
https://hdl.handle.net/2376/113608
PMID: 17207506
Abstract
Our mutagenesis and kinetic analyses of the large subunit of the potato AGPase heterotetramer suggest that the large subunit is essential not only for allosteric regulation but also for catalysis of the enzyme. The catalytic significance of the large subunit is further emphasized by photoaffinity labeling experiment with ATP analog which shows that the large subunit binds ATP as efficiently as the small subunit.
ADP-glucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis, is composed of a pair of catalytic small subunits (SSs) and a pair of catalytically disabled large subunits (LSs). The N-terminal region of the LS has been known to be essential for the allosteric regulatory properties of the heterotetrameric enzyme. To gain further insight on the role of this region and the LS itself in enzyme function, the six proline residues found in the N-terminal region of the potato tuber AGPase were subjected to scanning mutagenesis. The wildtype and various mutant heterotetramers were expressed using our newly developed host-vector system, purified, and their kinetic parameters assessed. While P
17L, P
26L, and P
55L mutations only moderately affected the kinetic properties, P
52L and P
66L gave rise to significant and contrasting changes in allosteric properties: P
66L enzyme displayed up-regulatory properties toward 3-PGA while the P
52L enzyme had down-regulatory properties. Unlike the other mutants, however, various mutations at P
44 led to only moderate changes in regulatory properties, but had severely impaired catalytic rates, apparent substrate affinities, and responsiveness to metabolic effectors, indicating Pro-44 or the LS is essential for optimal catalysis and activation of the AGPase heterotetramer. The catalytic importance of the LS is further supported by photoaffinity labeling studies, which revealed that the LS binds ATP at the same efficiency as the SS. These results indicate that the LS, although considered having no catalytic activity, may mimic many of the catalytic events undertaken by the SS and, thereby, influences net catalysis of the heterotetrameric enzyme.
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Details
- Title
- Catalytic implications of the higher plant ADP-glucose pyrophosphorylase large subunit
- Creators
- Seon-Kap Hwang - Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USAShigeki Hamada - Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USAThomas W Okita - Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA
- Publication Details
- Phytochemistry (Oxford), Vol.68(4), pp.464-477
- Academic Unit
- Biological Chemistry, Institute of
- Publisher
- Elsevier Ltd
- Identifiers
- 99900548131701842
- Language
- English
- Resource Type
- Journal article