A mutant of ubiquitin C-terminal hydrolase L1 (UCHL1) detected in early-onset neurodegenerative patients, UCHL1, showed higher catalytic activity than wild-type UCHL1 (UCHL1). Lying within the active-site pocket, the arginine is part of an interaction network that holds the catalytic histidine in an inactive arrangement. However, the structural basis and mechanism of enzymatic activation upon glutamine substitution was not understood. We combined X-ray crystallography, protein nuclear magnetic resonance (NMR) analysis, enzyme kinetics, covalent inhibition analysis, and biophysical measurements to delineate activating factors in the mutant. While the crystal structure of UCHL1 showed nearly the same arrangement of the catalytic residues and active-site pocket, the mutation caused extensive alteration in the chemical environment and dynamics of more than 30 residues, some as far as 15 Å away from the site of mutation. Significant broadening of backbone amide resonances in the HSQC spectra indicates considerable backbone dynamics changes in several residues, in agreement with solution small-angle X-ray scattering (SAXS) analyses which indicate an overall increase in protein flexibility. Enzyme kinetics show the activation is due to a k effect despite a slightly weakened substrate affinity. In line with this, the mutant shows a higher second-order rate constant (k/K) in a reaction with a substrate-derived irreversible inhibitor, Ub-VME, compared to the wild-type enzyme, an observation indicative of a more reactive catalytic cysteine in the mutant. Together, the observations underscore structural plasticity as a factor contributing to enzyme kinetic behavior which can be modulated through mutational effects.