The activation mechanism of human porphobilinogen synthase by 2-mercaptoethanol: intrasubunit transfer of a reserve zinc ion and coordination with three cysteines in the active center

Abstract

Human porphobilinogen synthase [EC.4.2.1.24] is a homo-octamer enzyme. In the active center of each subunit, four cysteines are titrated with 5,5′-dithiobis(2-nitrobenzoic acid). Cys¹²², Cys¹²⁴ and Cys¹³² are placed near two catalytic sites, Lys¹⁹⁹ and Lys²⁵², and coordinate a zinc ion, referred to as “a proximal zinc ion”, and Cys²²³ is placed at the orifice of the catalytic cavity and coordinates a zinc ion, referred to as “a distal zinc ion”, with His¹³¹ . When the wild-type enzymes C122A (Cys¹²²→Ala), C124A (Cys¹²⁴→Ala), C132A (Cys¹³²→Ala) and C223A (Cys²²³→Ala) were oxidized by hydrogen peroxide, the levels of activity were decreased. Two cysteines were titrated with 5,5′-dithiobis(2-nitrobenzoic acid) in the wild-type enzyme, while on the other hand, one cysteine was titrated in the mutant enzymes. When wild-type and mutant enzymes were reduced by 2-mercaptoethanol, the levels of activity were increased: four and three cysteines were titrated, respectively, suggesting that a disulfide bond was formed among Cys¹²², Cys¹²⁴ and Cys¹³² under oxidizing conditions. We analyzed the enzyme-bound zinc ion of these enzymes using inductively coupled plasma mass spectrometry with gel-filtration chromatography. The results for C223A showed that the number of proximal zinc ions correlated to the level of enzymatic activity. Furthermore, zinc-ion-free 2-mercaptoethanol increased the activity of the wild-type enzyme without a change in the total number of zinc ions, but C223A was not activated. These findings suggest that a distal zinc ion moved to the proximal binding site when a disulfide bond among Cys¹²², Cys¹²⁴ and Cys¹³² was reduced by reductants. Thus, in the catalytic functioning of the enzyme, the distal zinc ion does not directly contribute but serves rather as a reserve as the next proximal one that catalyzes the enzyme reaction. A redox change of the three cysteines in the active center accommodates the catch and release of the reserve distal zinc ion placed at the orifice of the catalytic cavity.