Inactivation of GDP-mannose dehydrogenase from Pseudomonas aeruginosa by penicillic acid identifies a critical active site loop.

The pathogenic bacterium Pseudomonas aeruginosa synthesizes alginate as one of a group of virulence factors that are produced during infections. The enzyme GDP-mannose dehydrogenase catalyzes the committed step in alginate biosynthesis. We show here that penicillic acid is an irreversible inactivator of GDP-mannose dehydrogenase. Inactivation occurs with a rate constant of 0.39+/-0.01 mM(-1) min(-1) at pH 8.0, and does not exhibit saturation behavior. Partial protection from inactivation is afforded by GDP-mannose, but not by the other substrate, NAD+. GMP and NAD+ together provide complete protection against inactivation. Analysis by mass spectrometry confirmed that the enzyme is alkylated at multiple cysteine residues by penicillic acid, including Cys 213, Cys 246, and the active site cysteine, Cys 268. However, the pH dependence of the inactivation rate suggested that alkylation of a single cysteine residue is sufficient to inactivate the enzyme. The C268A mutant protein was also susceptible to inactivation by penicillic acid. The presence of NAD+ and GMP provided partial protection of Cys 246 and Cys 268, and almost complete protection of Cys 213. Cys 213 is located on a helix that forms part of the binding pocket for GDP-mannose, and forms a hydrogen bond with Asn 252. Asn 252 is located on a loop that surrounds GDP-mannose. The C213A mutant enzyme exhibits a Vmax that is 1.8-fold greater than the wild-type enzyme, suggesting that the interaction between Cys 213 and Asn 252 helps to hold the loop in place during catalysis, and that opening the loop to release product is partially rate-limiting. Cys 246 is adjacent to the GDP-mannose binding loop, and its alkylation may also interfere with loop movement.

Disentangling the web of allosteric communication in a homotetramer: heterotropic inhibition of phosphofructokinase from Bacillus stearothermophilus.

A strategy for isolating each of the four potentially unique heterotropic pairwise allosteric interactions that exist in the homotetramer phosphofructokinase from Bacillus stearothermophilus is described. The strategy involves the construction of hybrid tetramers containing one wild-type subunit and three mutant subunits that have been modified to block binding of both the substrate, fructose 6-phosphate (Fru-6-P), and the allosteric inhibitor, phospho(enol)pyruvate (PEP). Each type of binding site occurs at a subunit interface, and mutations on either side of the interface have been identified that will greatly diminish binding at the respective site. Consequently, four different types of mutant subunits have been created, each containing a different active site and allosteric site modification. The corresponding 1:3 hybrids isolate a different pair of unmodified substrate and allosteric sites with a unique structural disposition located 22, 30, 32, and 45 A apart, respectively. The allosteric inhibition exhibited by the unmodified sites in each of these four hybrids has been quantitatively evaluated in terms of a coupling free energy. Each of the coupling free energies is unique in magnitude, and their relative magnitudes vary with pH. Importantly, the sum of these coupling free energies at each pH is equal to the total heterotropic coupling free energy associated with the tetrameric enzyme. The latter quantity was assessed from the overall inhibition of a control hybrid that removed the homotropic interactions in PEP binding. The results do not agree with either the concerted or sequential models that are often invoked to explain allosteric behavior in oligomeric enzymes.