Energetics of substrate binding and catalysis by class 1 (glycosylhydrolase family 47) alpha-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control.
Nascent glycoproteins are subject to quality control in the lumen of the endoplasmic reticulum (ER) where they can either be effectively folded with the aid of a collection of ER chaperones or they can be targeted for disposal in a process known as ER-associated degradation. Initiation of the ER disposal process involves selective trimming of N-glycans by ER alpha-mannosidase I and subsequent recognition by the ER degradation-enhancing alpha-mannosidase-like protein family of lectins, both members of glycosylhydrolase family 47. The kinetics and energetics of substrate binding and catalysis by members of this family were investigated here by the analysis of wild type and mutant forms of human ER alpha-mannosidase I. The contributions of several amino acid residues and an enzyme-associated Ca(2+) ion to substrate binding and catalysis were demonstrated by a combination of surface plasmon resonance and enzyme kinetic analyses. One mutant, E330Q, shown previously to alter general acid function within the catalytic site, resulted in an enzyme that possessed increased glycan binding affinity but compromised glycan hydrolysis. This mutant protein was used in a series of glycan binding studies with a library of mannose-containing ligands to examine the energetics of Man(9)GlcNAc(2) substrate interactions. These studies provide a framework for understanding the nature of the unusual substrate interactions within the family 47 mannosidases involved in glycan maturation and ER-associated glycoprotein degradation.
Karaveg
Khanita
K
Complex Carbohydrate Research Center and the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, 30602, USA.
Moremen
Kelley W
KW
D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011134:Polysaccharides / Q000378:metabolism*; D013696:Temperature; D043323:alpha-Mannosidase / Q000737:chemistry / Q000378:metabolism*
D006023:Glycoproteins; D011134:Polysaccharides; D043323:alpha-Mannosidase; D002118:Calcium