No abstract

Insights into the early molecular events involving protein-ligand/substrate interactions such as protein signaling and enzyme catalysis can be obtained by examining these processes on a very short, millisecond time scale. We have used time-resolved electrospray mass spectrometry to delineate the catalytic mechanism of a key enzyme in bacterial lipopolysaccharide biosynthesis, 3-deoxy-d-manno-2-octulosonate-8-phosphate synthase (KDO8PS). Direct real-time monitoring of the catalytic reaction under single enzyme turnover conditions reveals a novel hemiketal phosphate intermediate bound to the enzyme in a noncovalent complex that establishes the reaction pathway. This study illustrates the successful application of mass spectrometry to reveal transient biochemical processes and opens a new time domain that can provide detailed structural information of short-lived protein-ligand complexes.

The mechanism of oligomerization and its role in the regulation of activity in large GTPases are not clearly understood. Human guanylate binding proteins (hGBP-1 and 2) belonging to large GTPases have the unique feature of hydrolyzing GTP to a mixture of GDP and GMP with unequal ratios. Using a series of truncated and mutant proteins of hGBP-1, we identified a hydrophobic helix in the connecting region between the two domains that plays a critical role in dimerization and regulation of the GTPase activity. The fluorescence with 1-8-anilinonaphthalene sulfonate and circular dichroism measurements together suggest that in the absence of the substrate analog, the helix is masked inside the protein but becomes exposed through a substrate-induced conformational switch, and thus mediates dimerization. This is further supported by the intrinsic fluorescence experiment, where Leu(298) of this helix is replaced by a tryptophan. Remarkably, the enzyme exhibits differential GTPase activities depending on dimerization; a monomer produces only GDP, but a dimer gives both GDP and GMP with stimulation of the activity. An absolute dependence of GMP formation with dimerization demonstrates a cross talk between the monomers during the second hydrolysis. Similar to hGBP-1, hGBP-2 showed dimerization-related GTPase activity for GMP formation, indicating that this family of proteins follows a broadly similar mechanism for GTP hydrolysis.

SummaryArginase is a binuclear Mn 21 -metalloenzyme of urea cycle that catalyzes the conversion of L-arginine to L-ornithine and urea. Unlike other arginases, the Helicobacter pylori enzyme is selective for Co 21 , and has lower catalytic activity. To understand the differences in the biochemical properties as well as activity compared to other arginases, we carried out a detailed investigation of different metal reconstituted H. pylori arginases that includes steady-state kinetics, fluorescence measurement, pH-dependent and oligomerization assays. Unlike other arginases (except human at physiological pH), the Co 21 -and Mn 21 -reconstituted H. pylori enzymes exhibit cooperative mechanism of arginine hydrolysis, and undergo self-association and activation with increasing concentrations. Analytical gel-filtration assays in conjunction with the kinetic data showed that the protein exists as a mixture of monomer and dimer with monomer being the major form (other arginases exclusively exist as a trimer or hexamer) but the dimer is associated with higher catalytic activity. The proportion of dimer is found to decrease with increasing salt concentrations indicating that salt bridges play important roles in dimerization of the protein. Furthermore, the fluorescence measurement showed that Co 21 ions play an important role in the local tertiary structure of the protein than Mn 21 . This is consistent with the pH-dependent studies where the Co 21 -enzyme showed a single ionization compared to the double in the Mn 21 -enzyme. Thus, this study presents the detailed biochemical and spectroscopic investigations into the differences in the biochemical properties and activity between H. pylori and other arginases.