Mannosidase specificity

Mutant 1,2-a-D-mannosidase Specific activity mkatal/kga Relarive activity %... 

Swainsonine (1) is of great biochemical interest since it is a potent and specific inhibitor of both lysosomal a-mannosidase and mannosidase II, which are involved in the cellular degradation of polysaccharides and in the processing of asparagine-linked glycoproteins, respectively [1]. 

Y. D. Lobsanov, F. Vallee, A. Imberty, T. Yoshida, P. Yip, A. Herscovics, and P. L. Howell, Structure of Penicillium citrinum al,2-mannosidase reveals the basis for differences in specificity of the endoplasmatic reticulum and golgi class I enzymes, J. Biol. Chem., 277 (2002) 5620-5630. 

Investigation of the inhibitory effects on glycosidases has been carried out for the hydroxylated pyrrolidines [65] and piperidines [73]. Among the pyrrolidines prepared only the l,4-dideoxy-l,4-imino-L-allitol (23) (Scheme 7) and its C-5 isomer showed any remarkable effects. They inhibited lysosomal a-manno-sidase rather than the processing a-mannosidases I and II [65], and their specificity is in accord with the structural requirements of azafuranose analogues of mannose for inhibiting mammalian a-o-mannosidases [80]. 

Since the isolation of the chitinase inhibitor allosamidin from Streptomyces [90], the aminohydroxy-substituted cyclopentanes have been recognized as powerful and specific inhibitors of glycosidases [91]. The synthesis of (+) [92], as well as of racemic [93] mannostatin, which is a strong mannosidase inhibitor, should be mentioned here (Fig. 1). 

D-GIucal (l,5-anhydro-2-deoxy-D-ara/nno-hex-l-enitol) has been shown to be a competitive inhibitor of a-D-mannosidase,70 but it has less than one-tenth of the inhibitory power of D-mannono-1,5-lactone. D-Glucal is less specific than D-mannono-1,5-lactone, because it also inhibits a- and /3-D-glucosidases.71... 

To establish that a-D-mannosidase is a metalloenzyme, the proportion of bound zinc in the active molecule must first be determined. This experiment has been performed for the enzyme from jack-bean meal.27 Two types of preparation were employed. The first was purified, throughout, at pH 8 without any addition of Zn2+ (see Section 11,4 p. 409). The second enzyme preparation was purified in the presence of added Zn2+ at pH 5 up to stage 5 of the original procedure (see Table V p. 410). It was then freed from unbound Zn2+ by dialysis against glycine buffer of pH 8, and passed through a column of Sephadex G-100 in the same buffer. As may be seen from Table IX, the final specific activity was, in each instance, slightly less than that shown in Table V. 

As the Zn2+ required for catalytic activity could be retained during purification, a-D-mannosidase may be classified as a metalloenzyme. The enzyme seems to be completely specific for Zn2+ as the activating cation. 

Recovery of a-D-mannosidase (units" per g of meal) Recovery of Zn (fig per g of meal) Specific activity Metal content" fig per g of protein) ... 

In zinc-deficient rats, the zinc content of the epididymis was only about half the normal value,28,87 and the level of a-D-mannosidase activity was also little over half the value usually observed.26 However, the zinc concentration of the tissue was still in vast excess over that required for stoichiometric combination with the enzyme protein, calculated on any probable estimate of its specific activity and molecular weight (see Section III,5 p. 433). 

Enzymes. See also, Amylases, Galactosi-dases, /3-Glucuronidase, Glyco-enzymes, a-D-Mannosidase. acting on pectic substances, 5, 79-102 degradation by, of starch and glycogen, 3, 251-310 17, 407-430 of glycoprotein structure, 27, 301-341 specificity of, in the domain of carbohydrates, 5, 49-78... 

Inhibitors can block the pathway at specific steps, causing the accumulation of biosynthetic intermediates. Castanospermine inhibits the a-glucosidase that removes the first glucose deoxymannojirimycin inhibits the a-mannosidase I that removes mannose from the Man8 intermediate, and swainsonine blocks the a-mannosidase II that removes mannose from the Man5 intermediate. In the presence of swainsonine, hybrid N-linked carbohydrates are synthesized. 

The predicted amino acid sequence of the 1,2-a-D-mannosidase from A. saitoi (107) is 70, 26, and 35% identical with those of P. citrunum 1,2-a-D-mannosidase [108], yeast (S. cerevisiae) Man9-specific mannosidase [109, 110] and mouse Golgi 1,2-a-D-mannosidase from [111], respectively. 

Table 16. Specific activities of purified wild type and mutant 1,2-a-D-mannosidases from...

---