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  • The kinetic data obtained from incubation of BAB with GLO

    2022-09-09

    The kinetic data obtained from incubation of PYR-41 synthesis 4BAB with GLO1 show that 4BAB does not completely inactivate the enzyme (Figs. 2A and S6a). At low concentrations (4.7 and 14.4μM), 4BAB seemed to follow biphasic kinetics (Fig. 2A); even though regardless of the concentration 4BAB, there was a plateau at approximately the same level of maximal activity. This was quite unexpected as we had proposed that 4BAB enters the enzyme active site and orients the bromine near Cys60 for nucleophilic attack, which should completely abolished enzyme activity. This led to the question of how 4BAB could be covalently modifying GLO1 but result in partial enzyme inactivation. One explanation was that 4BAB could possibly be binding to GLO1 somewhere other than the enzyme active site, which results in partial loss of enzyme activity. However, this does not seem likely, since it was shown that the transition state analogue CHG protects GLO1 from inactivation by 4BAB (Fig. 2D), which suggests that inactivation is an active site directed process. Therefore, another molecular phenomenon must be causing the observed partial inactivation. Another possibility is that there is active GLO1 monomer present in the assay solution, and it is this species that inactivated by 4BAB leading to the observed activity decrease. However this most likely is not the case since the native PAGE shows that GLO1 (Fig. S5b) is present exclusively as the dimer. Furthermore, there are no reports to our knowledge of monomeric human GLO1 retaining enzyme activity. This led to the PYR-41 synthesis that only one active site of the GLO1 homodimer is being modified by 4BAB. To test this hypothesis, GLO1 treated with 4BAB was analyzed by LC–MS and two species were observed in the reaction mixture; one that corresponded to the unmodified GLO1 monomer and one that corresponded to GLO1 monomer with 4BAB (minus the bromine) attached. Furthermore, the two species are present in nearly equal quantities, although the peaks were not sufficiently resolved to integrate or quantify (Fig. S7). If there was only one active site of GLO1 modified, then it would be expected to maintain 50% of its activity but only 33% activity remains. We proposed that modification of one active site of GLO1 could cause a conformational change affecting the other active site present in the dimeric form. The kinetic constants were measured for GLO1 control and for the GLO1:4BAB complex (Fig. 2B, Table 1) and the kcat and Km values obtained for GLO1 are in good agreement with values previously reported for the enzyme. The GLO1:4BAB enzyme complex showed reduced catalytic efficiency such that there was a 2/3 loss of catalytic turnover (decreased kcat) and about 1/5 decrease in substrate binding (increased Km) for the GLO1:4BAB enzyme complex compared to the wild-type enzyme. This is consistent with the observation that GLO1 retains about 33% activity in the presence of 4BAB (Fig. 2A). A scenario could be envisioned in which one molecule of 4BAB binds covalently to a single active site resulting in complete inactivation simultaneously causing a conformational change around the opening to the hydrophobic pocket of second active site that results in the reduced kinetic parameters observed. This conformational change could presumably prevent a second molecule of 4BAB from orienting the bromine group near Cys60 of the second active site for attack. The inaccessibility to the hydrophobic pocket should not have that much of an effect on the binding of the thiohemiacetal substrate (about 20% decrease observed), since the methyl group of substrate is much smaller than the tail region of 4BAB. The proposal that modification at one active site affects the binding at the second active site seems contradictory to the results reported for bivalent transition state analogues, which have a lower Ki than the transition state molecules by themselves. However, these bivalent transition state analogues bind to the active site in the same manner as a single transition state molecule, which is by coordination of the N-hydroxycarbamoyl moiety of the molecule to the active site zinc ion. There has yet to be evidence that indicates that GLO1 has cooperative binding with regards to substrate or transition state analogues.34, 41 Allosteric coupling has been reported for the two active sites of the monomeric Plasmodium falciparum GLO1 and was proposed for the two active sites of monomeric yeast GLO1 but no reports of allosteric binding have been reported for the dimeric human enzyme.