Breaking the covalent connection: Chain connectivity and the catalytic reaction of PMM/PGM.
Fragment complementation has been used to investigate the role of chain connectivity in the catalytic reaction of phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa, a human pathogen. A heterodimer of PMM/PGM, created from fragments corresponding to its first three and fourth domains, was constructed and enzyme activity reconstituted. NMR spectra demonstrate that the fragment corresponding to the fourth (C-terminal) domain exists as a highly structured, independent folding domain, consistent with its varied conformation observed in enzyme-substrate complexes. Steady-state kinetics and thermodynamics studies reported here show that complete conformational freedom of Domain 4, because of the break in the polypeptide chain, is deleterious to catalytic efficiency primarily as a consequence of increased entropy. This extends observations from studies of the intact enzyme, which showed that the degree of flexibility of a hinge region is controlled by the precise sequence of amino acids optimized through evolutionary constraints. This work also sheds light on the functional advantage gained by combining separate folding domains into a single polypeptide chain.
Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA.
Andrew M
AM
Schramm
Dale
D
Karr
Ritcha
R
Mehra-Chaudhary
Steven R
SR
Van Doren
Cristina M
CM
Furdui
Lesa J
LJ
Beamer
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010733:Phosphoglucomutase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017875:Phosphotransferases (Phosphomutases) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D001426:Bacterial Proteins; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D017875:Phosphotransferases (Phosphomutases); D010733:Phosphoglucomutase; C052799:phosphomannomutase