The PROPKA method for the prediction of the pKa values of ionizable residues in proteins is extended to include the effect of non‐proteinaceous ligands on protein pKa values as well as predict the change in pKa values of ionizable groups on the ligand itself. This new version of PROPKA (PROPKA 2.0) is, as much as possible, developed by adapting the empirical rules underlying PROPKA 1.0 to ligand functional groups. Thus, the speed of PROPKA is retained, so that the pKa values of all ionizable groups are computed in a matter of seconds for most proteins. This adaptation is validated by comparing PROPKA 2.0 predictions to experimental data for 26 protein–ligand complexes including trypsin, thrombin, three pepsins, HIV‐1 protease, chymotrypsin, xylanase, hydroxynitrile lyase, and dihydrofolate reductase. For trypsin and thrombin, large protonation state changes (|n| > 0.5) have been observed experimentally for 4 out of 14 ligand complexes. PROPKA 2.0 and Klebe's PEOE approach (Czodrowski P et al. J Mol Biol 2007;367:1347–1356) both identify three of the four large protonation state changes. The protonation state changes due to plasmepsin II, cathepsin D and endothiapepsin binding to pepstatin are predicted to within 0.4 proton units at pH 6.5 and 7.0, respectively. The PROPKA 2.0 results indicate that structural changes due to ligand binding contribute significantly to the proton uptake/release, as do residues far away from the binding site, primarily due to the change in the local environment of a particular residue and hence the change in the local hydrogen bonding network. Overall the results suggest that PROPKA 2.0 provides a good description of the protein–ligand interactions that have an important effect on the pKa values of titratable groups, thereby permitting fast and accurate determination of the protonation states of key residues and ligand functional groups within the binding or active site of a protein. Proteins 2008. © 2008 Wiley‐Liss, Inc.

• We used molecular dynamics, saturation mutagenesis, and enzymologic screening to develop a glutaminase-free mutant (Q59L) L-ASP.• We then used Q59L to show that glutaminase activity is not required for L-ASP activity against ASNS-negative cancer cells.L-Asparaginase (L-ASP) is a key component of therapy for acute lymphoblastic leukemia. Its mechanism of action, however, is still poorly understood, in part because of its dual asparaginase and glutaminase activities. Here, we show that L-ASP's glutaminase activity is not always required for the enzyme's anticancer effect. We first used molecular dynamics simulations of the clinically standard Escherichia coli L-ASP to predict what mutated forms could be engineered to retain activity against asparagine but not glutamine. Dynamic mapping of enzyme substrate contacts identified Q59 as a promising mutagenesis target for that purpose. Saturation mutagenesis followed by enzymatic screening identified Q59L as a variant that retains asparaginase activity but shows undetectable glutaminase activity. Unlike wild-type L-ASP, Q59L is inactive against cancer cells that express measurable asparagine synthetase (ASNS). Q59L is potently active, however, against ASNS-negative cells. Those observations indicate that the glutaminase activity of L-ASP is necessary for anticancer activity against ASNS-positive cell types but not ASNS-negative cell types.Because the clinical toxicity of L-ASP is thought to stem from its glutaminase activity, these findings suggest the hypothesis that glutaminase-negative variants of L-ASP would provide larger therapeutic indices than wild-type L-ASP for ASNS-negative cancers. (Blood. 2014;123(23):3596-3606) Introduction L-Asparaginase (L-ASP) is an enzyme drug used in combination with vincristine and a glucocorticoid (eg, dexamethasone) to treat acute lymphoblastic leukemia (ALL).1,2 We 3-6 and others 7 have reported a rationale for testing L-ASP against low-asparagine synthetase (ASNS) solid tumors as well. L-ASP's primary known enzymatic activity is deamidation of asparagine to aspartic acid and ammonia, but it also deamidates glutamine to glutamic acid and ammonia, although with lower affinity and lower maximal rate. L-ASP therapy is often limited by toxic side effects that are generally attributed to the glutaminase activity. 8,9 Those side effects often preclude completion of the full treatment regimen, resulting in poor outcome. 10 The question that arises, however, is whether the therapeutic index of L-ASP could be increased by decreasing its glutaminase activity 8,9 or whether that would also decrease the anticancer effect commensurately.One side of the debate hypothesizes that L-ASP's therapeutic index can be improved by increasing glutaminase activity. In support of that hypothesis, data collected over the last decade have suggested that glutaminase activity generally increases the efficacy of L-ASP and is sometimes required to achieve an anticancer effect. Those studies have reported asparaginase activity to be expendable. [11][12][13][...

The diet of the snow leopard (Panthera uncia) was studied from 213 scats collected between April 1990 and February 1991 in the Annapurna Conservation Area. Nepal. Seven species of wild and five species of domestic mammals were taken, as well as an unidentified mammal and birds. Blue sheep (Pseudois nayaur) were the most frequently eaten prey. Himalayan marmots (Marmota himalayana) were also important, except in winter when they were hibernating. During winter, snow leopards ate more Royle's pika (Odiotona royki) and domestic livestock. Yaks were eaten more frequently than other livestock types.