Functional diversity among putative E2 isozymes in the mechanism of ubiquitin-histone ligation.

The covalent ligation of the 8.6-kDa protein ubiquitin to histones within transcriptionally poised regions is believed to participate in the localized regulation of chromatin structure. This unique post-translational modification is thought to be distinct from similar cytosolic reactions in requiring ubiquitin-activating enzyme (E1) and one or more putative ubiquitin carrier proteins (E2) but not isopeptide ligase (E3). Apparently homogeneous preparations of the E2 isozymes were tested for their ability to catalyze the E3-independent conjugation of ubiquitin to linker histone H1 and core histones H2A, H2B, H3, and H4 in the presence of catalytic amounts of E1. Significant rates of nonprocessive core histone monoubiquitination were catalyzed by the E2(14kDa), E2(20kDa), and E2(32kDa) isozymes but not by either E2(17kDa) or E2(24kDa). The former three E2 isozymes also supported slow rates of direct multiple ubiquitination to secondary ligation sites on the histones. Rate studies for the monoubiquitination of H2A and H2B revealed that: 1) E2(14kDa) catalyzed a second order reaction with respect to histone concentration; 2) E2(32kDa)-mediated ligation proceeded by hyperbolic kinetics, yielding Km values of 2.8 and 12 microM for H2A and H2B, respectively; and 3) E2(20kDa) exhibited complex kinetics composed of both second order and hyperbolic pathways, the latter having Km values of 0.83 and 1.5 microM for H2A and H2B, respectively. Pulse-chase kinetics suggested that both ubiquitin thiol esters formed to E2(20kDa) were catalytically competent in H2A ligation. The active E2 isozymes also catalyzed the processive multiple ubiquitination of calf thymus H1. Other rate studies determined that Kd values for binding of the active E2 species to E1 ternary complex were 0.1 nM for E2(14kDa), 0.4 nM for E2(32kDa), and 3.6 nM for E2(20kDa). The data indicate that E2(20kDa) and E2(32kDa) are specific but mechanistically distinct ligation enzymes responsible for the conjugation of ubiquitin to nucleosomal proteins.