Regulatory and Structural Properties of the Cyanobacterial ADPglucose Pyrophosphorylases

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The apparent affinity for the inhibitor, phosphate, decreased greater than 400-fold. Mutation of Lys-441 to glutamic acid showed even larger effects. When Lys-417 and Lys-455 on the large subunit were mutated to alanine, the phosphate inhibition was not altered and the apparent affinity for the activator decreased only 9-and 3-fold, respectively. Mutations of these residues to glutamic acid only decreased the affinity for the activator 12-and 5-fold, respectively. No significant changes were observed on other kinetic constants for the substrates ADP-Glc, pyrophosphate, and Mg 2؉ . These data indicate that Lys-404 and Lys-441 on the small subunit are more important for the regulation of the ADP-Glc pyrophosphorylase than their homologous residues in the large subunit.

mentioning

confidence: 99%

ADP-Glucose Pyrophosphorylase from Potato Tubers. Site-Directed Mutagenesis Studies of the Regulatory Sites1

Ballícora

Nesbitt

et al. 1998

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“…The enzyme from higher plants (Preiss, 1988(Preiss, , 1991, green algae (Sanwal and Preiss, 1967;Ball et al, 1991), and cyanobacteria (Iglesias et al, 1991) is mainly activated by 3PGA and inhibited by Pi. ADPGlc PPases from enteric bacteria are activated by Fru-1,6-bisP and inhibited by AMP (Preiss and Romeo, 1989).…”

mentioning

confidence: 99%

Mutagenesis of the Glucose-1-Phosphate-Binding Site of Potato Tuber ADP-Glucose Pyrophosphorylase1

Ballícora

Preiss

1998

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Lysine (Lys)-195 in the homotetrameric ADP-glucose pyrophosphorylase (ADPGlc PPase) from Escherichia coli was shown previously to be involved in the binding of the substrate glucose-1-phosphate (Glc-1-P). This residue is highly conserved in the ADPGlc PPase family. Site-directed mutagenesis was used to investigate the function of this conserved Lys residue in the large and small subunits of the heterotetrameric potato (Solanum tuberosum) tuber enzyme. The apparent affinity for Glc-1-P of the wild-type enzyme decreased 135-to 550-fold by changing Lys-198 of the small subunit to arginine, alanine, or glutamic acid, suggesting that both the charge and the size of this residue influence Glc-1-P binding. These mutations had little effect on the kinetic constants for the other substrates (ATP and Mg 2؉ or ADP-Glc and inorganic phosphate), activator (3-phosphoglycerate), inhibitor (inorganic phosphate), or on the thermal stability. Mutagenesis of the corresponding Lys (Lys-213) in the large subunit had no effect on the apparent affinity for Glc-1-P by substitution with arginine, alanine, or glutamic acid. A double mutant, S K198R L K213R , was also obtained that had a 100-fold reduction of the apparent affinity for Glc-1-P. The data indicate that Lys-198 in the small subunit is directly involved in the binding of Glc-1-P, whereas they appear to exclude a direct role of Lys-213 in the large subunit in the interaction with this substrate.

“…However, the purification and yield values are an overestimation because the heat treatment caused a 3.5-fold increase in total enzyme activity (Table I). Although this phenomenon was observed in other purifications (Plaxton and Preiss, 1987;Iglesias et al, 1991), the increases by the heat treatment were usually not significant (below 1.5-fold). We consistently observed a 3-to 5-fold increase in enzyme activity by the heat treatment step.…”

Section: Purification Of Agp From Tomato Fruitmentioning

confidence: 89%

“…We consistently observed a 3-to 5-fold increase in enzyme activity by the heat treatment step. As suggested by Iglesias et al (1991), this could be attributed to the presence of an inhibitor in the crude extract. Also, it is likely that interfering reactions occurred in the crude extract that lowered the enzyme activity values in that step.…”

Section: Purification Of Agp From Tomato Fruitmentioning

confidence: 97%

Multiple Forms of ADP-Glucose Pyrophosphorylase from Tomato Fruit

Chen

Janes

1997

ADP-glucose pyrophosphorylase (ACP) was purified from tomato (Lycopersicon esculentum Mill.) fruit to apparent homogeneity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme migrated as two close bands with molecular weights of 50,000 and 51,000. Two-dimensional polyacrylamide gel electrophoresis analysis of the purified enzyme, however, revealed at least five major protein spots that could be distinguished by their slight differences i n net charge and molecular weight. Whereas all of the spots were recognized by the antiserum raised against tomato fruit ACP holoenzyme, only three of them reacted strongly with the antiserum raised against the potato tuber ACP large subunit, and the other two spots (with lower molecular weights) reacted specifically with antisera raised against spinach leaf ACP holoenzyme and the potato tuber ACP small subunit. l h e results suggest the existente of at least three isoforms of the ACP large subunit and two isoforms of the small subunit in tomato fruit in vivo. The native molecular mass of the enzyme determined by gel filtration was 220 f 1 O kD, indicating a tetrameric structure for ACP from tomato fruit. l h e purified enzyme is very sensitive to 3-phosphoglycerate/ inorganic phosphate regulation.