Loss of activity of the lysosomal glycosidase β-glucocerebrosidase (GCase) causes the lysosomal storage disease Gaucher disease (GD) and has emerged as the greatest genetic risk factor for the development of both Parkinson disease (PD) and dementia with Lewy bodies. There is significant interest into how GCase dysfunction contributes to these diseases, however, progress toward a full understanding is complicated by presence of endogenous cellular factors that influence lysosomal GCase activity. Indeed, such factors are thought to contribute to the high degree of variable penetrance of
GBA
mutations among patients. Robust methods to quantitatively measure GCase activity within lysosomes are therefore needed to advance research in this area, as well as to develop clinical assays to monitor disease progression and assess GCase-directed therapeutics. Here, we report a selective fluorescence-quenched substrate, LysoFQ-GBA, which enables measuring endogenous levels of lysosomal GCase activity within living cells. LysoFQ-GBA is a sensitive tool for studying chemical or genetic perturbations of GCase activity using either fluorescence microscopy or flow cytometry. We validate the quantitative nature of measurements made with LysoFQ-GBA using various cell types and demonstrate that it accurately reports on both target engagement by GCase inhibitors and the
GBA
allele status of cells. Furthermore, through comparisons of GD, PD, and control patient-derived tissues, we show there is a close correlation in the lysosomal GCase activity within monocytes, neuronal progenitor cells, and neurons. Accordingly, analysis of clinical blood samples using LysoFQ-GBA may provide a surrogate marker of lysosomal GCase activity in neuronal tissue.
Glycoside hydrolases
(GHs) catalyze hydrolyses of glycoconjugates
in which the enzyme choreographs a series of conformational changes
during the catalytic cycle. As a result, some GH families, including
α-amylases (GH13), have their chemical steps concealed kinetically.
To address this issue for a GH13 enzyme, we prepared seven cyclohexenyl-based
carbasugars of α-d-glucopyranoside that we show are
good covalent inhibitors of a GH13 yeast α-glucosidase. The
linear free energy relationships between rate constants and pK
a of the leaving group are curved upward, which
is indicative of a change in mechanism, with the better leaving groups
reacting by an SN1 mechanism, while reaction rates for
the worse leaving groups are limited by a conformational change of
the Michaelis complex prior to a rapid SN2 reaction with
the enzymatic nucleophile. Five bicyclo[4.1.0]heptyl-based carbaglucoses
were tested with this enzyme, and our results are consistent with
pseudoglycosidic bond cleavage that occurs via SN1 transition
states that include nonproductive binding of the leaving group to
the enzyme. In total, we show that the conformationally orthogonal
reactions of these two carbasugars reveal mechanistic details hidden
by conformational changes that the Michaelis complex of the enzyme
and natural substrate undergoes which align the nucleophile for efficient
catalysis.
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