Transient kinetic investigation of GTP hydrolysis catalyzed by interferon-gamma-induced hGBP1 (human guanylate binding protein 1).
Within the family of large GTP-binding proteins, human guanylate binding protein 1 (hGBP1) belongs to a subgroup of interferon-inducible proteins. GTP hydrolysis activity of these proteins is much higher compared with members of other GTPase families and underlies mechanisms that are not understood. The large GTP-binding proteins form self-assemblies that lead to stimulation of the catalytic activity. The unique result of GTP hydrolysis catalyzed by hGBP1 is GDP and GMP. We investigated this reaction mechanism by transient kinetic methods using radioactively labeled GTP as well as fluorescent probes. Substrate binding and formation of the hGBP1 homodimer are fast as no lag phase is observed in the time courses of GTP hydrolysis. Instead, multiple turnover experiments show a rapid burst of P(i) formation prior to the steady state phase, indicating a rate-limiting step after GTP cleavage. Both molecules are catalytically active and cleave off a phosphate ion in the first step. Then bifurcation into catalytic inactivation, probably by irreversible dissociation of the dimer, and into GDP hydrolysis is observed. The second cleavage step is even faster than the first step, implying a rapid rearrangement of the nucleotide within the catalytic center of hGBP1. We could also show that the release of the products, including the phosphate ions, is fast and not limiting the steady state activity. We suggest that slow dissociation of the GMP-bound homodimer gives rise to the burst behavior and controls the steady state. The assembled forms of the GDP- and GMP-bound states of hGBP1 are accessible only through GTP binding and hydrolysis and achieve a lifetime of a few seconds.
Kunzelmann
Simone
S
Physikalische Chemie 1, Fakultät für Chemie und Biochemie, Ruhr-Universität Bochum, 44780 Bochum, Germany.
Praefcke
Gerrit J K
GJ
Herrmann
Christian
C
D002384:Catalysis; D020134:Catalytic Domain; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D007371:Interferon-gamma / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D013696:Temperature
C490287:GBP1 protein, human; D006153:Guanosine Diphosphate; D007371:Interferon-gamma; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins