Energetics of Substrate Binding and Catalysis by Class 1 (Glycosylhydrolase Family 47) α-Mannosidases Involved in N-Glycan Processing and Endoplasmic Reticulum Quality Control

Khanita Karaveg,Kelley W Moremen

doi:10.1074/jbc.m505130200

Abstract

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.

Highlights

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
Kinetic Analysis of ER ␣-mannosidase I (ERManI) Mutants—Mutations were generated previously in five residues that were hypothesized to be involved in catalysis by ERManI [46]. These mutations tested the roles of putative general acid (E330Q and R334A) and general base (E599Q and H524A) functions, as well as a residue that proved to play a critical role in providing hydrogen bonding interactions with the mannose residue in the ϩ1 subsite (D463N) (Fig. 1, C and D)
Distortion of the glycone into a 3S1 conformation during substrate binding has been proposed to predispose the substrate for hydrolysis by a least motion conformational twist through a ringflattened 3H4 transition state producing an inverted enzymatic product in a 1C4 conformation [46]

Summary

Introduction

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. For terminally misfolded glycoproteins, trimming of the oligosaccharide by the action of ER ␣-mannosidase I (ERManI) to generate a unique Man8GlcNAc2 isomer product (Fig. 1E) is the key rate-limiting initiation signal [9, 10] that leads to retrotranslocation of the polypeptide back into the cytoplasm for degradation by the proteasome in a process known as ER-associated degradation (ERAD) [11]. The present models envisage recognition of the glycan structures by the EDEM proteins in a mode similar to substrate recognition during catalysis by the true hydrolases, followed by transfer to the Sec translocon pore, retrotranslocation into the cytosol, and proteasomal degradation [2] Understanding how this family of enzymes and lectins accomplish their functions in recognition and catalysis will provide insights into the rate-

Methods

Results

Conclusion