Structural Biochemistry/GTPase

Overview

GTPases are a large family consisting of hydrolase enzymes that bind and hydrolyze guanosine triphosphate (GTP). Binding and hydrolysis of GTP occurs in a region common to all GTPases, the highly conserved G domain.

Function

• GTPases play a major role in a several functions:
• Signal transduction in the intracellular space of transmembrane receptors (taste, smell, and light)
• Protein translation in the ribosome
• Regulation during cell division, translocation of proteins through membranes
• Transportation and control of assembly of vesicles in the cell.

Mechanism

Nucleophilic substitution, specifically SN2, is the mechanism for how the hydrolysis of the γ phosphate in GTP convert it into GDP and an inorganic phosphate (Pi). The mechanism also involves a pentavalent intermediate and a magnesium ion (Mg2+).

Superfamilies

There are several "superfamilies" within the family of GTPases.

Regulatory GTPases

Regulatory GTPases are GTPases that are responsible for the regulation of biochemical processes. G proteins are the most prominent of the superfamily.

GTP Switch Mechanism

All regulatory GTPases have a common mechanism that allows them to "switch" a signal transduction chain on and off. GTPase-activating proteins, or GTPase-accelerating proteins (GAPs), are a family of regulatory proteins that can bind to active G-proteins and stimulate their GTPase activity. They usually come from another signal transduction chain. The binding of GAPs to GTPases stimulates the change of GTPase between its two forms and this is what causes the toggling of the "switch" for the signal transduction chain. GTP-bound is the active form and when it gets hydrolyzed to its inactive GDP-bound form, GTPase is consequently inactivated. Of course, this inactivation can be reverted by Guanine nucleotide exchange factors (GEFs). GEFs activate GTPases by changing it back to the active GTP-bound form via initiation of the release of GDP from GTPase to allow binding to a new GTP molecule. It is important to note that the hydrolysis of GTP to GDP is irreversible and this causes the cycle to the active GTP-bound form of GTPase to be closed. Only active GTPases can produce a signal to a new reaction chain.

The efficiency of signal transduction via active GTPase is dependent on the active to inactive ratio of GTPase:

${\frac {{\mbox{GTPase}}*{\mbox{GTP}}}{{\mbox{GTPase}}*{\mbox{GDP}}}}={\frac {k_{\mbox{diss.GDP}}}{k_{\mbox{cat.GTP}}}}$

There are two constants that can be modified by special regulatory proteins: kdiss.GDP = dissociation constant of GDP kcat.GTP = hydrolysis constant of GTP

The amount of active GTPase can be modified in several ways:

• Acceleration of GDP dissociation by GEFs significantly speeds up the construction of active GTP-bound GTPase
• Acceleration of GTP hydrolysis by GAPs reduces the amount of active GTP-bound GTPase
• Inhibition of GDP dissociation by Guanine nucleotide dissociation inhibitors (GDIs)slows down construction of active GTP-bound GTPase. (GDIs bind to small Rho and Rab GDP-bound GTPases and keep the GTPase inactivated, as well as preventing the GTPase from localizing at their place of action, their membrane.)
• GTP analogues that cannot be hydrolyzed (γ-S-GTP, β,γ-methylene-GTP, and β,γ-imino-GTP) can keep GTPase active.

Ras GTPases/Small GTPases

Ras GTPases, or small GTPases, serve as switches for cell signalling. They get their names from a family of proteins called the Ras subfamily, which are proteins that are involved in cell signal transmission, because these GTPases are small monomeric proteins that are homologous to Ras proteins. Additionally, the Ras GTPases can be split up into 5 subcategories:

• Ras
• Rho
• Rab
• Ran
• Arf

Intracellular Membrane Traffic

Members of the Rab and Arf branches of the Ras GTPase superfamily are present in every step of intracellular membrane traffic. They regulate these steps by networking with one another through a variety of mechanisms that coordinate independent events of one stage together with other stages of the entire transport pathway. These mechanisms include many different variables:

• GEFs cascades
• GAPs cascades
• effectors that bind many GTPases
• positive feedback loops stemming from exchange factor-effector interactions.

When these mechanisms come together, an ordered series of transitions from one GTPase to the next can take place. Since each GTPase has its own unique group of effectors, the transitions that occur can help define differences in the functionality of the membrane compartments that they are associated with.

Large GTPases--Dynamin

Dynamin is a considered model for large GTPases. It is responsible for endoctyosis, a process in which cells absorb molecules by engulfment. Specifically, it is involved in the division of newly formed vesicles from the membrane of one compartment to their fusion with another compartment-- at both the cell surface or Golgi body. Along with division of vesicles, Dynamin is also involved in the division of organelles, cytokinesis, and pathogen resistance (microbial). In mammals, there are 3 different types of genes:

• Dynamin I: expressed in most cells
• Dynamin II: expressed in neurons
• Dynamin III: expressed in heart, brain, lung, and testis.

Mechanism

When a vesicle folds in so that the outer surface becomes an inner surface, dynamin will form a spiral around the vesicle's neck. The spiral will then extend and then constrict via GTP hydrolysis. This process produces a twisting motion and results in the pinching off of the vesicle from its main body. The twisting motion is dependent on its dynamin GTPase activity. So far, dynamin is the only right-handed helix that produces a twisting motion (right-handed twisting).

Translation Factor GTPases

Translation Factor GTPases have an important role in the initiation, elongation, and the termination of protein biosynthesis.

References

1. Mizuno-Yamasaki, E., F. Rivera-Molina, and et al. "GTPase networks in membrane traffic.." Pub Med. N.p., 29 2012. Web. 7 Dec 2012. <http://www.ncbi.nlm.nih.gov/pubmed/22463690>.

2. "GTPase." Wikipedia. Wikimedia Foundation, Inc. 28 May 2012. Web. 7 Dec 2012. <http://en.wikipedia.org/wiki/GTPase>.

3. "Guanine nucleotide exchange factor." Wikipedia. Wikimedia Foundation, Inc. 6 Jun 2012. Web. 7 Dec 2012. <http://en.wikipedia.org/wiki/Guanine_nucleotide _exchange_factor>.

4. "Guanosine nucleotide dissociation inhibitors." Wikipedia. Wikimedia Foundation, Inc. 21 Mar 2012. Web. 7 Dec 2012. <http://en.wikipedia.org/wiki/Guanosine_nucleotide_dissociation_inhibitors>.

5. "GTPase-activating protein." Wikipedia. Wikimedia Foundation, Inc. 4 Nov 2012. Web. 7 Dec 2012. <http://en.wikipedia.org/wiki/GTPase-activating_ protein>.

6. "Ras subfamily." Wikipedia. Wikimedia Foundation, Inc. 1 Nov 2012. Web. 7 Dec 2012. <http://en.wikipedia.org/wiki/Ras_(protein)>.

7. "Endocytosis." Wikipedia. Wikimedia Foundation, Inc. 6 Dec 2012. Web. 7 Dec 2012. <http://en.wikipedia.org/wiki/Endocytosis>.

8. "Dynamin." Wikipedia. Wikimedia Foundation, Inc. 2 Apr 2012. Web. 7 Dec 2012. <http://en.wikipedia.org/wiki/Dynamin>.