An active site loop mutant of a zinc metallo-deubiquitinase suggests the importance of loop dynamics to catalysis

Bueno, Amy N. M.S., Purdue University, May 2015. An Active Site Mutant of a Zinc Metallo‐Deubiquitinase Suggests the Importance of Loop Dynamics to Catalysis. Major Professor: Chittaranjan Das. AMSH is a conserved zinc metalloprotease that functions with endosomal sorting complexes required for transport (ESCRT) machinery in order to down regulate and degrade cell‐surface receptors. It has high specificity toward Lys63‐linked polyubiquitin chains, a signal used for receptor down regulation by the ESCRT machinery. It has previously been found that Sst2, an AMSH orthologue from fission yeast, contains a flexible “loop”, referred to herein as the active‐site flap, near the proximal ubiquitin binding site, composed of Leu402 and Phe403. Mobility of the side chains of these residues contributes to the opening and closing conformation of the catalytic cleft which may play important role in catalysis. The dynamics of the active‐site flap may also contribute to linkage specificity. To gain insight into the role played by the active‐site flap in catalysis, point mutants of the conserved Phe are generated. Reported herein are studies of a Trp mutant (Phe403Trp) of Sst2 using X‐ray crystallography, enzyme kinetics, isothermal calorimetry (ITC), and circular dichroism (CD) which indicate the importance of dynamics of the flap in catalysis and substrate selectivity of the enzyme.