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

A L Haas,P M Bright,V E Jackson

doi:10.1016/s0021-9258(18)37700-7

Abstract

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.

Highlights

Which shows a 10-fold lower apparent affinity for activating Three lines of evidence indicate that both thiol ester sites enzyme, since no discontinuity in the reciprocal plot for E l dependence was observed (Fig. 3), as would have been expected were the data theresult of tight complex formation
On E 2 m k ~p~articipate in histone ligation, either directly or these E2 isozymes, which exhibited little processivity since indirectly: 1) the sum of the first order rate constants for the respective monoubiquitinated species were the predomichase of 1251-ubiquitin fromboth thiol ester sites on E220k~a nant adducts, Fig. 1
(0.6 min”) agreed with the katvalue calculated directly from multiple ubiquitination are not intrinsic to a given E 2 isozyme the initial rate of uH2A formation; 2) the first order plot for but ratherare highly dependent on the identityof the histone chase of label fromthe dithiol ester band of E 2 2 0 k r~e~mained substrate

Summary

Introduction

Which shows a 10-fold lower apparent affinity for activating Three lines of evidence indicate that both thiol ester sites enzyme, since no discontinuity in the reciprocal plot for E l dependence was observed (Fig. 3), as would have been expected were the data theresult of tight complex formation.

Results

Conclusion