Abstract
Glycosidases are widespread in nature, where they perform a diverse range of functions. The glycoside hydrolase (GH) family 38, α-mannosidase II enzymes play a crucial role in mammalian cells, in the maturation of N-glycosylated proteins in the Golgi apparatus and in catabolism in cytosol and lysosomes of glycans originating from e.g. terminally misfolded proteins. A divalent metal ion typically resides in the active site, and is essential for enzyme activity. Recently, the α-mannosidase II from Sulfolobus solfataricus (ManA) was purified and characterized as a Zn2+-containing enzyme, but also displayed metal ion promiscuity. The present thesis (Manuscript 1) describes the steady state kinetics of ManA in complex with various activating divalent metal ions and substrates. The Co2+-enzyme was superior on both para-nitrophenyl-α-Dmannoside and on 1,2-, 1,3-, 1,4- and 1,6-α-mannobiose. On para-nitrophenyl-α-Dmannoside the Co2+ substituted ManA displayed a 6-fold higher kcat/KM compared to ManA substituted with Cd2+ and Zn2+. On the α-mannobioses the difference was even more pronounced, with the Co2+substituted ManA being the only metal ion enzyme complex, which allowed the determination of individual KM and kcat values on all α-mannobioses. With Cd2+ and Zn2+, ManA appeared highly inefficient while ManA substituted with Mn2+, contrary to that observed on para-nitrophenyl-α-D-mannoside, displayed a relatively high activity on 1,2- and 1,4-α-mannobiose. The binding mechanism of ManA obeys a steady state ordered mechanism, in which the metal ion must bind prior to the substrate. Ni2+ and Cu2+ were inhibitors of ManA, which probably bind to the active site, but result in an enzyme unable to facilitate substrate binding.
Numerous glycosidases have a carbohydrate binding domain (CBM) appended to the
catalytic domain. CBMs are predominantly thought to maintain the enzyme on its
substrate to artificially increase the substrate concentration. Analogously, secondary
binding sites (SBSs), which, contrary to CBMs are located on the catalytic domain,
have been suggested to perform a similar task. Many putative SBSs have been identified in various enzymes, often based on crystal structures, and only few have been characterized in terms of structure-function relationship. Together SBS1 and SBS2 of barley α-amylase isozyme 1 probably represent the two most extensively studied SBSs. SBS2, largely governed by Tyr380, has been shown to be important for AMY1 adhesion to starch granules, but seems to play no significant role in the degradation of oligosaccharides, and only a minor role in the degradation of amylose. In Manuscript 2, a steady state kinetic analysis of amylopectin depolymerization by AMY1 and the SBS2 impaired mutant enzyme Y380A is described, in an effort to determine the potential role of SBS2 in amylopectin degradation. Progress curves of amylopectin degradation was best described by a bi-exponential equation comprising two rate constants (for reaction 'a' and 'b') associated with the degradation of 2 different parts of the amylopectin molecule, represented by 40% and 60%, respectively. Saturation curves revealed a relatively low apparent KM for the 'a'-reaction and a relatively high KM for the 'b'-reaction. ß-cyclodextrin, which has been shown to bind SBS2 and therefore compete with substrate binding, mainly inhibited the 'a'-reaction suggesting a role of SBS in the 'a'-reaction. In contrast Y380A mutant enzyme did not display a similarly low apparent KM for the 'a'-reaction, and inhibition by ß-cyclodextrin was not observed. In conclusion, SBS2 appears to play a role in amylopectin degradation by artificially decreasing the apparent KM for the 'a'-reaction.
Numerous glycosidases have a carbohydrate binding domain (CBM) appended to the
catalytic domain. CBMs are predominantly thought to maintain the enzyme on its
substrate to artificially increase the substrate concentration. Analogously, secondary
binding sites (SBSs), which, contrary to CBMs are located on the catalytic domain,
have been suggested to perform a similar task. Many putative SBSs have been identified in various enzymes, often based on crystal structures, and only few have been characterized in terms of structure-function relationship. Together SBS1 and SBS2 of barley α-amylase isozyme 1 probably represent the two most extensively studied SBSs. SBS2, largely governed by Tyr380, has been shown to be important for AMY1 adhesion to starch granules, but seems to play no significant role in the degradation of oligosaccharides, and only a minor role in the degradation of amylose. In Manuscript 2, a steady state kinetic analysis of amylopectin depolymerization by AMY1 and the SBS2 impaired mutant enzyme Y380A is described, in an effort to determine the potential role of SBS2 in amylopectin degradation. Progress curves of amylopectin degradation was best described by a bi-exponential equation comprising two rate constants (for reaction 'a' and 'b') associated with the degradation of 2 different parts of the amylopectin molecule, represented by 40% and 60%, respectively. Saturation curves revealed a relatively low apparent KM for the 'a'-reaction and a relatively high KM for the 'b'-reaction. ß-cyclodextrin, which has been shown to bind SBS2 and therefore compete with substrate binding, mainly inhibited the 'a'-reaction suggesting a role of SBS in the 'a'-reaction. In contrast Y380A mutant enzyme did not display a similarly low apparent KM for the 'a'-reaction, and inhibition by ß-cyclodextrin was not observed. In conclusion, SBS2 appears to play a role in amylopectin degradation by artificially decreasing the apparent KM for the 'a'-reaction.
| Original language | English |
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| Publisher | Department of Chemistry, Faculty of Science, University of Copenhagen |
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| Number of pages | 165 |
| Publication status | Published - 2012 |