The N-terminal Segment of Recombinant Porcine Fructose-1,6-bisphosphatase Participates in the Allosteric Regulation of Catalysis

Date
2001-03-01
Authors
Nelson, Scott
Kurbanov, Feruz
Nelson, Scott
Honzatko, Richard
Fromm, Herbert
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Abstract

Residues 1–10 of porcine fructose-1,6-bisphosphatase (FBPase) are poorly ordered or are in different conformations, sensitive to the state of ligation of the enzyme. Deletion of the first 10 residues of FBPase reducesk cat by 30-fold and Mg2+ affinity by 20-fold and eliminates cooperativity in Mg2+ activation. Although a fluorescent analogue of AMP binds with high affinity to the truncated enzyme, AMP itself potently inhibits only 50% of the enzyme activity. Additional inhibition occurs only when the concentration of AMP exceeds 10 mM. Deletion of the first seven residues reduces k cat and Mg2+ affinity significantly but has no effect on AMP inhibition. The mutation of Asp9 to alanine reproduces the weakened affinity for Mg2+ observed in the deletion mutants, and the mutation of Ile10 to aspartate reproduces the AMP inhibition of the 10-residue deletion mutant. Changes in the relative stability of the known conformational states for loop 52–72, in response to changes in the quaternary structure of FBPase, can account for the phenomena above. Some aspects of the proposed model may be relevant to all forms of FBPase, including the thioredoxin-regulated FBPase from the chloroplast.

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<p>This article is from <em>Journal of Biological Chemistry</em> 276 (2001): 6119, doi:<a href="http://dx.doi.org/10.1074/jbc.M009485200" target="_blank">10.1074/jbc.M009485200</a>. Posted with permission.</p>
Keywords
Biocatalysts, Enzyme inhibition, Magnesium printing plates, fructose bisphosphatase, isoleucine, thioredoxin, enzyme kinetics
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