Search

The kinetic behavior of chitinase A1 from Bacillus circulans WL-12 was investigated using the novel fluorogenic substrates, N-deacetylated 4-methylumbelliferyl chitobiosides [GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)(2)-UMB (4)], and the results were compared with those obtained using 4-methylumbelliferyl N, N'-diacetylchitobiose [(GlcNAc)(2)-UMB (1)] as the substrate. The chitinase did not release the UMB moiety from compound 4, but successfully released UMB from the other substrates. k(cat)/K(m) values determined from the releasing rate of the UMB moiety were: 145.3 for 1, 8.3 for 2, and 0.1 s(-1) M(-1) for 3. The lack of an N-acetyl group at subsite (-1) reduced the activity to a level 0.1% of that obtained with compound 1, while the absence of the N-acetyl group at subsite (-2) reduced the relative activity to 5.7%. These observations strongly support the theory that chitinase A1 catalysis occurs via a 'substrate-assisted' mechanism. Using these novel fluorogenic substrates, we were able to quantitatively evaluate the recognition specificity of subsite (-2) toward the N-acetyl group of the substrate sugar residue. The (-2) subsite of chitinase A1 was found to specifically recognize an N-acetylated sugar residue, but this specificity was not as strict as that found in subsite (-1).

Oligosaccharide binding to chitosanase from Streptomyces sp. N174 was indirectly evaluated from thermal unfolding experiments of the protein. Thermal unfolding curves were obtained by fluorescence spectroscopy in the presence of d -glucosamine oligosaccharides ((GlcN) n , n =3,4,5, and 6) using the inactive mutant chitosanase in which the catalytic residue, Glu 22 , is mutated to glutamine (E22Q), aspartic acid (E22D), or alanine (E22A). The midpoint temperature of the unfolding transition ( T m ) of E22Q was found to be 44.4°C at pH 7.0. However, the T m increased upon the addition of (GlcN) n by 1.3°C ( n =3), 2.5°C ( n =4), 5.2°C ( n =5), or 7.6°C ( n =6). No appreciable change in T m was observed when (GlcNAc) 6 was added to E22Q. The effect of (GlcN) n on the thermal stability was examined using the other protein, RNase T1, but the oligosaccharide did not affect T m of the protein. Thus, we concluded that the stabilization effect of (GlcN) n on the chitosanase results from specific binding of the oligosaccharides to the substrate binding cleft. When E22D or E22A was used instead of E22Q, the increases in T m induced by (GlcN) 6 binding were 2.7°C for E22D and 4.2°C for E22A. In E22D or E22A, interaction with (GlcN) 6 seems to be partly disrupted by a conformational distortion in the catalytic cleft.

Fucosyloligosaccharides have great therapeutic potential. Here we present a new route for synthesizing a Fucalpha1,2Gal linkage by introducing glycosynthase technology into 1,2-alpha-l-fucosidase. The enzyme adopts a unique reaction mechanism, in which asparagine-423 activated by aspartic acid-766 acts as a base while asparagine-421 fixes both a catalytic water and glutamic acid-566 (an acid) in the proper orientations. Glycosynthase activity of N421G, N423G, and D766G mutants was examined using beta-fucosyl fluoride and lactose, and among them, the D766G mutant most effectively synthesized 2'-fucosyllactose. 1,2-alpha-l-Fucosynthase is the first glycosynthase derived from an inverting alpha-glycosidase and from a glycosidase with an unusual reaction mechanism.

The hydrolytic reaction of family 18 chitinase has been considered to occur via substrate assisted catalysis. To kinetically investigate the enzyme reaction mechanism, we synthesized compounds designed to reduce the polarization of the carbonyl in N-acetyl group, GlcNAc-GlcN(TFA)-UMB (2) and GlcNAc-GlcN(TAc)-UMB (3). Kinetic parameters in the hydrolysis of these compounds by chitinase A from Serratia marcescens (ChiA) were compared with those from the hydrolysis of (GlcNAc)2-UMB (1). The kcat of 2 was 3.4% of 1, but the Km of 2 was 10-fold that of 1. In contrast, the kcat of 3 was only 0.3% of that of 1, and the two reactions had an identical Km. The drastic decreases in kcat were probably due to the weak nucleophilic activity of the C2-N-trifluoroacetamide and N-thioacetamide groups at reducing ends of compounds 2 and 3, respectively. These results indicate that the anchimeric assistance of the C2 N-acetamide group at GlcNAc plays a key role in the hydrolytic reactions catalyzed by family 18 chitinases.