QM/MM study of Guanosine triphosphate (GTP) hydrolysis in homologous enzymes

Recently, Swhwartz's group [1] reported their study on two bacterial enzymes EcMTAN and VcMTAN, in addition to the inherent interest in the function of these enzymes, they are also interesting test cases of how very similiar enzymes with experimentally determined transition states that are essentailly identical seem to have different reaction mechanisms, since they are reported to have different binding affinities for the same transition state analogue. So reaction coordiante information is crucial to explain, and it is only accessible from transition path sampling approaches, since all experimental approaches report only average.

This could also be the case for Guanosine triphosphate (GTP) hydrolysis in their homologous enzymes. With different participation of differnt enzyme environment, the GTP hydrolysis can happen in different ways, even though the transition state are somehow analogue, and the reaction mechanism in general is the same.

Phosphoryl transfer reactions play a crucial role in widespread cellular functions, ranging from the signaling [2] and stress-activated [3] pathways to the biosynthesis of nucleic acids [4]. The key step of action of dephosphorylating enzymes is the transfer of a phosporyl group of GTP to acceptors such as water (hydolases), amino acids residues (kinases) and other nucleotides (nucleoside monophosphate kinases). Within the family of hydrolases, the low molecular weight GTP-binding proteins (LMWGs) [5] hold a prominent position.

                              GTP + H2O → GDP + HPO4 ⁼

One member of this family is Cdc42/Cdc42GAP/GDP (PDB code 1GRN). Different and conflicting proposals for the enzymatic mechanism have been reported. They differ in three key points, which are also the common points for GTP/GDP enzymatic reaction study:

(1) the formation of the highly nucleophilic agent OH-; (2) its binding to GTP with the formation of GDP and inorganic phosphate. (3) dissociative or associative pathway in step (2)?

Conclusion: (1) H2O transfers its proton to Gln61, thus the highly nucleophilic OH- is stabilized. Meantime, low-barrier hydrogen bond formed between Lys16 and β-phosphate. (2) a chemical bond formed between γ-phosphate and WAT oxygen. (3) the reactant is pre-built as a dissociative reaction model.

Another important member of this family is the Elongation factor Tu (EF-Tu). There is a “star” amino acid that often under debates is the histidine 85 (His85), who locates in the switch II region. One point of view is that His85 acts as a base, it participates directly to the OH- formation [6], the contrary is that His85 is not a base, but only contribute to the stabilization of the transition state by hydrogen binding [7]. However, it is proved that the replacement of His84 with Ala reduces the rate constant of GTP hydrolysis more than 10⁶ - fold. But not yet any fix conclusion of its role made. 

There is one interesting article from Nemukhin’s group [8] who talked about two cases: His85 in (side chain of His85 approach the reaction active site) and His85 out (side chain of His85 stays away from the active site). The reactions’ transition and product states were shown in Figure 2.

 

Conclusion: The character of TS is closer to the dissociative type reaction mechanism than to the associative type. In the His85 in case, His85 serves as a general base, while the His85 out case, the reaction results as a consequence of proton transfer mediated by two water molecules. The His85 in case show much lower activation barrier which corresponds to experimental result. But it remains still a interesting study point.

References:

[1] Matthew, I. Z.; Motley, M. W. ; Antoniou, D. ; Schramm V. L . ; Schwartz S. D. J. Phys. Chem. B. 2015, 119, 3662-3668

[2] Takai, Y.; Kishimoto, A.; Inoue, M.; Nishizuka, Y. J. Biol. Chem. 1977, 252, 7603-7609

[3] Kyriakis, J. M.: Avruch, J. J. Biol. Chem. 1996, 271, 24313-24316

[4] Koerner, J. F. Annu. Rev. Biochem. 1970, 39, 291-322.

[5] Shinjo, K.; Koland, J. G.; Hart, M. J.; Narasimhan, V.; Johnson, D. I.; Evans, T.; Cerione, R. A. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 9853-9857

[6] Voorhees, M. R; Schmeing, T. M; Kelley, A. C; Ramakrishnan, K. V. Science 2010, 330, 835

[7] Daviter, T; Wieden, H; Rodnina, M. V. J. Mol. Biol. 2003, 332, 689.

[8] Grigorenko, B.L.; Shadrina, M.S.; Topol I.A.; Collins J.R.; Nemukhin A.V. Bichimica et Biophysica Acta 2008, 1784, 1908-1917