Single-molecule studies reveal method for tuning the heterogeneous activity of Alkaline Phosphatase

Biophys J. 2022 May 7:S0006-3495(22)00372-1. doi: 10.1016/j.bpj.2022.05.005. Online ahead of print.ABSTRACTSingle-molecule enzymology (SME) methods have enabled the observation of heterogeneous catalytic activities within a single enzyme population. Heterogenous activity is hypothesized to originate from conformational changes in the enzyme that result from changes in the local environment leading to catalytically-active substates. Here, we use SME to investigate the mechanisms of heterogenous activity exhibited by tissue nonspecific alkaline phosphatase (TNSALP), which reveals two subpopulations with different catalytic activities. We show the effect of pH and temperature on the distribution of TNSALP activity and confirm the presence of two subpopulations attributed to half-active and fully-active TNSALP substates. We provide mechanistic insight about protein structure using molecular dynamic simulations and show pH- and temperature-dependent conformational transitions that corroborate experimentally observed changes in TNSALP activity. These results show the utility of SME to understand heterogeneous enzyme activity and demonstrate a simple approach using pH and temperature to tune catalytic activity within an enzyme population.PMID:35527401 | DOI:10.1016/j.bpj.2022.05.005
Source: Biophysical Journal - Category: Physics Authors: Source Type: research