Ubiquitin adenylate: structure and role in ubiquitin activation

Abstract

The acid precipitate of the ubiquitin activating enzyme after reaction with ATP and ubiquitin contains one enzyme equivalent of ubiquitin adenylate in which the carboxyl-terminal glycine of ubiquitin and AMP are in an acyl-phosphate linkage. The recovered ubiquitin adenylate has the catalytic properties proposed for it as a reaction intermediate. Thus, upon reaction with fresh enzyme in the absence of Mg2+ or ATP, the product complex, E-ubiquitin . AMP-ubiquitin, is formed. This complex is capable of generating ubiquitin-protein isopeptide derivatives when added to a reticulocyte fraction that catalyzes protein conjugation. This reproduces the effect previously shown to require ubiquitin, ATP, and Mg2+. In the presence of activating enzyme, ubiquitin adenylate is converted to ATP and free ubiquitin in a step requiring PPi and Mg2+. On the basis of studies of [32P]PPi/nucleoside triphosphate exchange, the activating enzyme could be used to generate 2'-deoxy-AMP-, 2'-deoxy-IMP-, and 2'-deoxy-GMP-ubiquitin but not pyrimidine nucleotide-ubiquitin derivatives. The enzyme shows a modest preference for the pro-S diastereomers of adenosine 5'-O-(1-thiotriphosphate) and adenosine 5'-O-(2-thiotriphosphate). Inorganic phosphate, arsenate, methyl phosphate, and tripolyphosphate, but not nucleoside triphosphates, can serve as alternate substrates in place of PPi in the reverse of ubiquitin adenylate formation. Therefore, the enzyme catalyzes the unusual reaction ATP + Pi in equilibrium ADP + PPi in the presence of ubiquitin.

Conservation in the Mechanism of Nedd8 Activation by the Human AppBp1-Uba3 Heterodimer

“…Affinity Purification of Human AppBp1-Uba3 Heterodimer from Human Erythrocytes-The AppBp1-Uba3 complex was isolated from human red blood cell Fraction II using Nedd8 affinity chromatography by adapting earlier methods for the isolation of Uba1 (28,37). Recombinant Nedd8 was coupled to Affi-Gel 10 (Bio-Rad) at ϳ0.5 mg of Nedd8/ml of resin for a final concentration of 60 M Nedd8 (37).…”

“…Stoichiometric Assays for Active AppBp1-Uba3 Heterodimer-The stoichiometric formation of covalent Nedd8 [ 3 H]adenylate was measured directly using [2, H]ATP as described previously for ubiquitinactivating enzyme (28,29). Fifty-l assays containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , 5 M Nedd8, 2 mg/ml carrier bovine serum albumin, 0.5 IU inorganic pyrophosphatase, 0.5 M [2, H]ATP (35,200 cpm/pmol), and about 1 pmol of AppBp1-Uba3 heterodimer were incubated at 37°C for 5 min.…”

“…In the activation of ubiquitin, Uba1 forms a ternary complex composed of 1 eq each of a tightly bound ubiquitin adenylate and a covalent Uba1-ubiquitin thiol ester to a conserved active site, Cys 632 (28,29), HsUba1a numbering. Early ATP:PP i exchange studies demonstrated that rabbit Uba1 catalyzes an absolutely ordered mechanism in which ATP binding precedes that of ubiquitin prior to the first catalytic step of ubiquitin adenylate formation (29).…”

“…Early ATP:PP i exchange studies demonstrated that rabbit Uba1 catalyzes an absolutely ordered mechanism in which ATP binding precedes that of ubiquitin prior to the first catalytic step of ubiquitin adenylate formation (29). The activated ubiquitin moiety is subsequently transferred to the thiol ester site comprising Cys 632 prior to formation of a second ubiquitin adenylate, to generate the final ternary complex (28,29). Marked conservation among the activating enzymes for ubiquitin and ubiquitinlike proteins argues that they proceed by similar mechanisms.…”

Intracellular protein catabolism: state of the art

“…The conjugation of ubiquitin to substrate proteins takes place via the formation of an adenylated ubiquitin catalysed by a ubiquitin-activating enzyme (El) and subsequently the formation of an El-ubiquitin thiol ester bond. The process results in the hydrolysis of ATP [87,88]. The transfer of ubiquitin to substrate proteins is catalyzed by enzyme E3 which transfers the ubiquitin moiety from another intermediate (E2-S-Ub) [89].…”