Recent developments in single-molecule enzymology (SME) have allowed for the observation of subpopulations present in enzyme ensembles. Tissue-nonspecific alkaline phosphatase (TNSALP), a homodimeric monophosphate esterase central to bone metabolism, has become a model enzyme for SME studies. TNSALP contains two internal disulfide bonds that are critical for its effective dimerization; mutations in its disulfide bonding framework have been reported in patients with hypophosphatasia, a rare disease characterized by impaired bone and tooth mineralization. In this paper, we present the kinetics of these mutants and show that these disulfide bonds are not crucial for TNSALP enzymatic function. This surprising result reveals that the enzyme's active conformation does not rely on its disulfide bonds. We posit that the signs and symptoms seen in hypophosphatasia are likely not primarily due to impaired enzyme function, but rather decreased enzyme expression and trafficking.
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