X-ray crystallography has revealed two similar alpha/beta domains of the aspartate racemase from the hyperthermophilic archaeon, Pyrococcus horikoshii OT3. The active site is located in the cleft between the two domains where two cysteine residues face each other. This arrangement allows the substrate to enter the cleft and enables the two cysteine residues to act synergistically. However, the distance between their thiolates was estimated to be 9.6 angstroms, which is beyond the distance for cooperative action of them. We examined the molecular mechanism for the racemization reaction of this hyperthermophilic aspartate racemase by mutational analyses and molecular dynamics simulations. The mutational analyses revealed that Arg48 and Lys164 were essential for catalysis in addition to the putative catalytic cysteine residues. The molecular dynamics simulations revealed that the distance between the two active gamma-sulfur atoms of cysteine residues oscillate to periodically become shorter than the predicted cooperative distance at high temperature. In addition, the conformation of Tyr160, which is located at the entrance of the cleft and inhibits the entry of a substrate, changes periodically to open the entrance at 375 K. The opening of the gate is likely to be induced by the motion of the adjacent amino acid, Lys164. The entrance of an aspartate molecule was observed by molecular dynamics (MD) simulations driven by the force of the electrostatic interaction with Arg48, Lys164, and also Asp47. These results provide insights into the roles of amino acid residues at the catalytic site and also the activation mechanism of a hyperthermophilic aspartate racemase at high temperature.

Objectives This subgroup analysis of the randomized, double-blind, Phase 3 ADVOCATE study evaluated the efficacy and safety of avacopan compared with tapered prednisone in Japanese patients with antineutrophil cytoplasmic antibody-associated vasculitis. Methods Patients with microscopic polyangiitis (MPA) or granulomatosis with polyangiitis (GPA) received either avacopan 30 mg twice daily for 52 weeks plus prednisone-matching placebo or tapered prednisone over 20 weeks plus avacopan-matching placebo for 52 weeks. The two primary efficacy endpoints were clinical remission at Week 26 and sustained remission at Week 52. Results Compared with the overall population (N = 330), Japanese patients (N = 21) were older and had worse renal function, and a higher proportion were female and had MPA. The proportion of Japanese patients with clinical remission at Week 26 was 9/11 (81.8%) with avacopan vs. 7/10 (70.0%) with prednisone (overall population: 72.3% vs. 70.1%) and with sustained remission at Week 52 was 8/11 (72.7%) vs. 4/10 (40.0%), respectively (overall population: 65.7% vs. 54.9%). The safety profile of avacopan was similar in Japanese patients and the overall study population. Conclusions The efficacy and safety of avacopan in Japanese patients with MPA or GPA were comparable to that observed in the overall ADVOCATE study population.

The neutral subtilopeptidase amylosacchariticus, a zinc metalloprotease, obtained from Bacillus subtilis var. amylosacchariticus was treated with chemical reagents which were known to modify tyrosyl residues of proteins. Acetylation of about 14 of 23 tyrosine residues of the enzyme with acetylimidazole led to almost complete losses of activities toward casein and synthetic peptide, Z‐Gly‐L‐Leu. NH2, and the activities lost were fully restored by the additional treatment with hydroxylamine. lodination of about three tyrosine residues also caused almost complete loss in the caseinolytic activity but increased the activity toward Z‐Gly‐L‐Leu. NH2 threefold over that of the native enzyme. Quite similar results were obtained when about three tyrosine residues were nitrated with tetranitromethane. Kinetic analysis indicated that Km value for Z‐Gly‐L‐Leu. NH2 remained approximately unchanged with the native, acetylated, iodinated, and nitrated enzymes, while KCat(Kmax/E) varied markedly depending upon the type of modification and the number of tyrosyl residues modified, increasing with nitration and iodination but decreasing with acetylation of tyrosyl residues. These results suggest that some tyrosine residues together with a zinc atom are closely related to the enzyme action.

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