Arginase is a bimetallic enzyme that utilizes mainly Mn 2+ or Co 2+ for catalytic function. In human homolog, substitution of Mn 2+ with Co 2+ significantly reduces the K m value without affecting the k cat . However, in the Helicobacter pylori counterpart (important for pathogenesis), the k cat increases nearly 4-fold with Co 2+ ions both in the recombinant holoenzyme and arginase isolated from H. pylori grown with Co 2+ or Mn 2+ . This suggests that the active site of arginase in the two homologs is modulated differently by these two metal ions. To investigate the underlying mechanism for metal-induced difference in catalytic activity in the H. pylori enzyme, we used biochemical, biophysical and microsecond MD simulations studies. The study shows that the difference in binding affinity of Co 2+ and Mn 2+ ions with the protein is linked to a different positioning of a loop (-- 122 HTAYDSDSKHIHG 134 --) that contains a conserved catalytic His133. Consequently, the proximity of His133 and conserved Glu281 is varied. We found that the Glu281-His133 interaction is crucial for catalytic function and was previously unexplored in other homologs. We suggest that the proximity difference between these two residues in the Co 2+ - and Mn 2+ -proteins alters the proportion of protonated His133 via variation in its p K a . This affects the efficiency of proton transfer-an essential step of L-arginine hydrolysis reaction catalyzed by arginase and thus activity. Unlike in human arginase, the flexibility of the above segment observed in H. pylori homolog suggests that this region in the H. pylori enzyme may be explored to design its specific inhibitors.
Interferon-γ inducible human guanylate binding protein-1 (hGBP1) shows a unique characteristic that hydrolyses GTP to a mixture of GDP and GMP through successive cleavages, with GMP being the major product. Like other large GTPases, hGBP1 undergoes oligomerization upon substrate hydrolysis, which is essential for the stimulation of activity. It also exhibits antiviral activity against many viruses including hepatitis C. However, which oligomeric form is responsible for the stimulated activity leading to enhanced GMP formation and its influence on antiviral activity, are not properly understood. Using mutant and truncated proteins, our data indicate that transition-state-induced tetramerization is associated with higher rate of GMP formation. This is supported by chimaeras that are defective in both tetramerization and enhanced GMP formation. Unlike wild-type protein, chimaeras did not show allosteric interactions, indicating that tetramerization and enhanced GMP formation are allosterically coupled. Hence, we propose that after the cleavage of the first phosphoanhydride bond GDP·P i -bound protein dimers transiently associate to form a tetramer that acts as an allosteric switch for higher rate of GMP formation. Biochemical and biophysical studies reveal that sequential conformational changes and interdomain communications regulate tetramer formation via dimer. Our studies also show that overexpression of the mutants, defective in tetramer formation in Rep2a cells do not inhibit proliferation of hepatitis C virus, indicating critical role of a tetramer in the antiviral activity. Thus, the present study not only highlights the importance of hGBP1 tetramer in stimulated GMP formation, but also demonstrates its role in the antiviral activity against hepatitis C virus.