Takeharu Masaki

Studies on a new proteolytic enzyme from Achromobacter lyticus M497-1. I. Purification and some enzymatic properties

Achromobacter lyticus M497-1 produces three kinds of alkaline proteases (protease I, II and III) in culture medium along with the bacteriolytic enzyme (Masaki, T., Nakamura, K., Isono, M. and Soejima, M. (1978) Agric. Biol. Chem. 42, 1443--1445). Among these three proteases, Achromobacter protease I (EC 3.4.21.-) shows strict splitting for lysine residues at the carboxyl side of the splitting point. This enzyme was purified through a sequence of benzalkonium chloride treatment, acetone fractionation, CM-cellulose and DEAE-cellulose treatment chromatography on AH-Sepharose 4B and isoelectric focusing method. This form was shown to be homogeneous by polyacrylamide gel electrophoresis and ultracentrifugation analysis. The physicochemical properties of the enzyme were: Mr 30 500; partial specific volume (v), 0.717 ml/g; intrinsic viscosity (nu), 0.0385) dl/g; isoelectric point (pI) 6.9; and E1%1cm at 280 nm, 18.77. The enzyme was composed of 294 residues of amino acid per molecule, with glycine as NH2-terminal and lysine as COOH-terminal amino acids. The optimum pH values with casein, Bz-lys-pNA and Tos-Lys-OMe were 8.5--10.7, 9.0--9.5 and 7.8--8.2, respectively. The enzyme was inhibited by iPr2P-F, PhCH2SO2F and Tos-LysCH2Cl but not by Tos-ArgCH2Cl, EDTA, o-phenanthroline and PCMB.

Studies on a new proteolytic enzyme from Achromobacter lyticus M497-1 II. Specificity and inhibition studies of Achromobacter protease I☆

The unique specificity of Achromobacter protease I for lysine residue was investigated using synthetic and natural substrates, i.e., lysine derivatives, arginine derivatives, lysine vasopressin, substance P, ACTH and insulin. The enzyme cleaved only the -Lys-X- bonds in the above substrates. The binding affinity of alkylamines as determined by Ki was much stronger than that of the corresponding alkylguanidines.

Purification, Bacteriolytic Activity, and Specificity of .BETA.-Lytic Protease from Lysobacter sp. IB-9374.

Lysobacter sp. IB-9374, which was isolated from soil as a high lysyl endopeptidase-producing strain (Chohnanet al., FEMS Microbiol. Lett., 213, 13-20, 2002), was found to produce a beta-lytic protease capable of lysing gram-positive bacteria such as Staphylococcus aureus, Microccocuseus, and Bacillus subtilis. The Lysobacter strain secreted the beta-lytic protease into the culture medium at a 2.4-fold higher level than Achromobacter lyticus. The enzyme was highly purified through a series of six steps with a high yield. The enzyme was strongly inhibited by tetraethylene-pentamine and 1,10-phenanthroline. The purified enzyme lysed more efficiently almost all the gram-positive bacteria tested than lysozyme, lysostaphin, and mutanolysin. The enzyme was very similar to Achromobacter beta-lytic protease containing one zinc atom in terms of amino acid composition and N-terminal sequence. The nucleotide sequence revealed that the mature enzyme was composed of 179 amino acid residues with additional 198 amino acids at the amino-terminal end of the enzyme. The deduced amino acid sequence of the mature enzyme coincided with that of the Achromobacter enzyme, although the prepro-region showed a 41% sequence identity with the counterpart. These results indicate that Lysobacter sp. is a useful strain for an efficient large-scale preparation of beta-lytic protease capable of lysing bacteria.

Neutron and X-ray crystallographic analysis of Achromobacter protease I at pD 8.0: Protonation states and hydration structure in the free-form

The structure of the free-form of Achromobacter protease I (API) at pD 8.0 was refined by simultaneous use of single crystal X-ray and neutron diffraction data sets to investigate the protonation states of key catalytic residues of the serine protease. Occupancy refinement of the catalytic triad in the active site of API free-form showed that ca. 30% of the imidazole ring of H57 and ca. 70% of the hydroxyl group of S194 were deuterated. This observation indicates that a major fraction of S194 is protonated in the absence of a substrate. The protonation state of the catalytic triad in API was compared with the bovine β-trypsin-BPTI complex. The comparison led to the hypothesis that close contact of a substrate with S194 could lower the acidity of its hydroxyl group, thereby allowing H57 to extract the hydrogen from the hydroxyl group of S194. H210, which is a residue specific to API, does not form a hydrogen bond with the catalytic triad residue D113. Instead, H210 forms a hydrogen bond network with S176, H177 and a water molecule. The close proximity of the bulky, hydrophobic residue W169 may protect this hydrogen bond network, and this protection may stabilize the function of API over a wide pH range.

Single-column separation of aminoethylcysteine other amino acids

Abstract Analytical conditions for aminoethylcysteine were established with the use of a single-column amino acid analyser equipped with a 150 x 4 mm I.D. column packed with Hitachi custom ion-exchange resin 2617 (5 μm). Using three buffer solutions twenty amino acids including aminoethylcysteine were separated for analysis. This method was applied to the acid hydrolysate produced by aminoethylation of insulin and lysozyme. The results showed that the method is effective not only for the determination of cysteine but also for acquiring better quantitative data on other amino acids.

A preliminary neutron diffraction analysis of Achromobacter protease I

Achromobacter protease I (API, E.C. 3.4.21.50) is one of the serine proteases produced by Achromobacter lyticus M497-1. API is distinct from the other tripsin type protease in its lysine specificity. The neutron structure analysis of catalytic triad with Trp169 and His210 was presented. His57 was double protonated and formed hydrogen bonds to Ser194Oγ and Asp113Oδ1, Oδ2.

Function of the carboxyl group specificity in Achromobacter protease I.

AchromobacterプロテアーゼIの活性中心部位,とくに基質特異性決定部位の性状を解明する手掛りを得る目的で,縮合剤と求核試薬との酵素活性に対する影響,強力な拮抗阻害剤であるω-アミノアルキル基をリガンドとするアフィニティクロマトグラフィーについて実験し,トリプシンと比較検討した. (1)本酵素はEDC単独,または求核試薬(エチレンジアミン)存在下でのEDCによって,トリプシンよりも弱いが明らかに阻害された.また拮抗阻害剤ブチルアミンで酵素を予備処理した後, EDCと求核試薬エチレンジアミンを反応させ,さらに透析することにより不活性化速度が減少した. (2)本酵素のアミノ基を無水酢酸でアセチル化しても,ほとんど酵素活性は影響されなかった. (3)炭素鎖を異にするω-アミノアルキル基をリガンドとするセファロース4B (Seph-NH-(CH2)n-NH2)に対する親和力をトリプシンと比較した結果,本酵素はメチレン基数が増大するとともに次第に強く吸着され, nが8で最大となり, nが8以上ではほぼ同様の強さで吸着された.また本酵素をTos-Lys CH2ClまたはiPr2P-Fによって完全に失活させた酵素はアミノオクチル(AO)セファロース4Bに吸着されなかった.同様に強力な拮抗阻害剤アミルアミン共存下で同様のクロマトグラフィーを行ったが吸着されなかった.