2 April 2007
Biol 6312
Catalysis (continued)
Cofactors
Cofactors are a diverse collection of small molecules that assist catalysis by enzymes at their active sites. They include metal ions, and small organic molecules usually called coenzymes. In humans most coenzymes are derived from vitamins. (Fig. 2-37)
Coenzymes often are also derived from ribonucleotides, suggesting they come from an RNA-only world that preceeded the current protein world.
The function of a coenzyme is to transfer a particular chemical group.
Metal ion cofactors are typically common elements in the earth's crust, and first row transition metals. (Fig. 2-38)
Some coenzymes are the products of chemical reactions between side chains of amino acids in proteins. One example is the green or red fluorescent protein. (Although not a true enzyme). Another is the LTQ or lysine tyrosylquinone found in copper amine oxidase. (Fig. 2-39).
Multi-Step Reactions
Many enzymes use multi-step reactions. A well-known example is the serine protease mechanism. One advantage is that several intermediates might each have lower transition states than a single step would. A difficult reaction is divided into simpler steps.
In the serine protease mechanism (Fig. 2-40):
Step 1: the serine -OH is a nucleophile. It is activated by proximity and orientation, and also by H-bonding to the N of the neighboring histidine. This breaks the peptide bond but makes an ester with the enzyme's Ser. The negative charge in the transition state to the intermediate is stabilized by 2 NH groups from the backbone.
Step 2: When the first product dissociates, water can move in to attack the ester. An ester is easier to break than an amide. The same transition state stabilization can occur in this step.
Phosphoryl transfer reactions also use a two-step mechanism.
In the first step a phosphoryl group is transfered to a residue of the enzyme. (It could be an-OH, -COOH, -SH or N of histidine). In the second step the phosphoryl is transfered to the substrate. An example of phosphoglucomutase is shown (Fig. 2-41)
Multifunctional Enzymes
There are 3 types of multifunctional enzymes:
Enzymes with a single active site that carry out more than one reaction.
Enzymes with 2 or more active sites.
Enzymes with 2 or more actives sites that are connected by an internal tunnel.
In the first case, generally, the reaction proceeds through a relatively stable intermediate, which inevitably proceeds to the next step, due to the arrangement of catalytic groups at the single active site. Example, isocitrate dehydrogenase (Fig. 2-42) In the first reaction, isocitrate is oxidized to oxalosuccinate using NADP+ (or NAD+). In the second reaction, oxalosuccinate is decarboxylated to alpha-ketoglutarate.
In the second case, the usual example is an enzyme with 2 domains, each of which contains an active site. Often, the product of one reaction is a substrate for the other active site. There does not appear to be interactions between the sites: the product must dissociate, and then diffuse to the next active site. The significance might be that the two active sites can be genetically regulated together. Typically, a bifunctional enzyme will not be bifunctional in all species. In Leishmania major, a parasite, thymidylate synthase and dihydrofolate reductase are encoded by a single polypeptide, whereas most organisms have two separate enzymes.
2'-deoxyUMP + N5,N10-methylene-tetrahydrofolate → thymidylate (TMP) + dihydrofolate
dihydrofolate + NADH → tetrahydrofolate + NAD
Another example: in purine biosynthesis ATIC (Fig. 2-43)
AICAR transformylase-IMP cyclohydrolase
AICAR + N10-formyl-tetrahydrofolate → FAICAR + tetrahydrofolate, and
FAICAR → IMP
AICAR is 5-aminoimidazole-4-carboxamide-ribonucleotide
FAICAR is formyl-5-aminoimidazole-4-carboxamide-ribonucleotide
IMP is inosine 5'-monophosphate, a purine precursor to adenine and guanine
In most species this is a bifunctional enzyme, but not in all.
Multifunctional Enzymes with Tunnels
This is the third, and small, class of multi-functional enzymes.
In this case the active sites are linked by a physical tunnel within the protein. It forces the products to move from one active site to another, without escaping into solution. This might be important for several different reasons. In one case, the product might diffuse out of the cell, for example, if it is uncharged, small, or nonpolar. Or the product might be unstable in aqueous solution.
First example, tryptophan synthase. A 25Å channel connects the two active site, allowing indole to diffuse from one to the other. In the first reaction, indole is cleaved from indole-3-glycerophosphate. In the second, it reacts with serine to generate tryptophan. (Fig. 2-44)
Another example is carbamoyl phosphate synthetase, which is a trifunctional enzyme. This is a single polypeptide with 3 active sites connected by 2 tunnels. The entire length is nearly 100 Å. (Fig. 2-45) Ammonia travels the first section. Carbamate travels the second section.
site 1: glutamine → glutamate + ammonia
site 2: ATP + bicarbonate → ADP + carboxy phosphate
ammonia + carboxy phosphate → carbamate + phosphate
site 3: carbamate + ATP → ADP + carbamoyl phosphate
Some enzymes have domains with non-enzyme functions
These functions include roles in regulation of transcription or translation, or signalling pathways.
Mechanisms of Regulation
Protein Interaction Domains
The interactions of many proteins are mediated by modular interaction domains. These domains are typically 35-150 amino acid residues in length. The N- and C- termini are usually close in space, allowing them to be accommodated as insertions into surface regions of almost any other fold. They can be categorized according to sequence, structure or ligand-binding properties.
(Fig. 3-2)
14-3-3 WD40 EF Hand LRR ARM SNARE PTB DD ANKYRIN C2 FHA BH SH2 SH3 PH SAM Bromo PDZ GYF Chromo FYVE RING finger WW LIM F-box C1 Fibronectin
Effector Ligands
Ligands that bring about effects on proteins through their binding can range in size from protons to proteins. Proton binding is usually considered a special class, and is discussed under the topic of pH effects. Protein binding is another special class discussed above. In between in size, are the so-called small molecules. They are often metabolites.
Competitive binding
This is a common situation, in which a product serves as an inhibitor of a reaction by binding at the catalytic site, i.e., it competes with the substrate. Almost all products can compete with the substrates of the the enzyme that produced them. Some also inhibit earlier enzymes in a metabolic pathway. That is called feedback inhibition. (Fig. 3-7)
Cooperativity
Cooperativity in binding of a ligand can occur if there are multiple subunits of the protein. The binding of one ligand by the protein influences the binding of subsequent ligands. In positive cooperativity, the subsequent ligands bind with enhanced affinity. In negative cooperativity, the subsequent ligands bind with diminished affinity. An extreme form of negative cooperativity is called "half-of-the-sites". In that case, binding of one ligand prevents binding of the second. In general, cooperativity is a consequence of the inherent flexibility of proteins, resulting in altered binding sites. (Fig. 3-8)
Allostery
Allostery is the situation when the effector ligand is different from the functional ligand. In this case the binding of the effector ligand at one site influences the affinity of the functional ligand at a different site. Allostery occurs because the binding of an effector ligand changes the conformation of the protein. This can occur through a sequence of conformational states (Fig. 3-9a) or through an equilibirium beween two symmetrical states (Fig. 3-9b). An effector molecule that increases affinity for a functional ligannd is called an allosteric activator. On that decreases affinity for a functional ligand is called an allosteric inhibitor.
Aspartate transcarbamoylase is an example of an allosteric enzyme. It has 6 catalytic subunits arranged in 2 trimers, and 6 regulatory subunits arranged in 3 dimers. There are 2 conformatonal states: a low-activity T state, in which regulatory subunits interact with the catalytic sites to decrease activity, and a high-activity R state in which the subunits have moved apart. (Fig. 3-10). CTP is an allosteric inhibitor. It is also a feedback inhibitor, since this enzyme catalyzes an early, but committed, step in pyrimidine synthesis. Its binding stabilizes the T state. ATP is an allosteric activator. This allows pyrimidine synthesis to catch up with purine synthesis. ATP stabilizes the R state of the enzyme. Both allosteric effectors and substrates bind cooperatively.
This enzyme illustrates the important point that an enzyme can be inhibited by the binding of a molecule at a site distant from the active site. Such effects can often be mimicked by mutations. Therefore, mutations that eliminate function should not be assumed to occur only at active sites.
Small molecules or ions that bind to activators or repressors of gene expression are known as co-activators or co-repressors. They can be thought of as allosteric effectors also. For example, Fe2+ binding is necessary for the diphtheria toxin repressor to dimerize properly to bind to the major groove of DNA. (Fig. 3-11)
Protein switches based on nucleotide hydrolysis
Many processes in cells are turned on or off by proteins that acts as molecular switches. The most common element in such proteins is that they undergo a conformational change based on the binding of nucleoside-diphosphates or nucleoside-triphosphates. Often the nucleotide used is guanosine triphosphate (GTP), and its hydrolysis product GDP. They have been termed G proteins. A second major class includes motor proteins that use ATP. These 2 classes have different folds, but bind the nucleotides in a similar way. (Fig. 3-12) This includes the "P-loop", which wraps around the phosphate region of the nucleotide, and the 2 "switch" regions, which undergo the significant changes in conformation. A schematic view of a G protein (Fig. 3-13)
The GTP-bound form of the protein is in the "on" state. It remains activated until the GTP is hydrolyzed, and the phosphate leaves. This triggers the conformational change to the GDP-bound form, which is the "off" state.
Signaling by small GTPases
An example of a monomeric, small GTPase protein is ras, part of a signal transduction pathway that is commonly found to be mutated to the "on" state in cancer.
The transition from the "off" state to the "on" state is facilitated by additional proteins called guanine-nucleotide exchange factors (GEFs). They bind to the GDP-bound form of the G-protein and cause release of the GDP. GTP will rapidly bind, causing a net exchange of GTP for GDP. (Fig. 3-14)
There might be several different GEFs that activate a single G-protein.
The transition from the "on" state to the "off" state depends upon the rate of GTP hydrolysis. In G-proteins, this rate is typically slow. That causes the protein to stay in the activated state for a long time. The rate can be increased by GTPase-activating proteins (GAPs). Again, several GAPs might activate a single G-protein. Some GAPs activate the GTPase by contributing residues to the active site, and contribute to transition-state stabilization. One example in the case of ras is the "arginine-finger", which helps to stabilize the negative charge that develops in the transition state.
Signaling by heterotrimeric GTPases
The other common class of G-proteins are heterotimeric: an α subunit that resemble the ras protein, and 2 tightly interacting proteins β and γ. They function with G-protein coupled receptors (GPCRs) at the cytoplasmic surface of the plasma membrane of eukaryotes. GCPRs are membrane proteins with 7 transmembrane spans, such as rhodopsin (Fig. 3-15)
Reaction cycle of heterotrimeric G-proteins:
The heterotrimer is bound to the surface of the plasma membrane in the GDP-bound or "off" state. This involves covalent prenylation of the α subunit. When activated, for example by absorbing a photon in the case of rhodopsin, the GCPR serves as a GEF to cause release of the GDP, and binding of GTP to the α subunit. This step requires that β and γ subunits be present.
After nucleotide exchange, the heterotrimer dissociates from the GCPR, and β and γ dissociate from α. They go on to take part in other signaling pathways. After GTP hydrolysis, the α subunit can rebind β and γ and then rebind to the GCPR.
The rate of GTP hydrolysis by heterotrimeric GTPases is stimulated by proteins called regulators of G-protein signaling (RGPS) proteins. Rather than contributing an arginine to the α subunit (which already has one), these proteins generally bind to the switch regions.
GTPases in protein synthesis
Elongation factors that deliver charged-tRNAs to the ribosome for protein synthesis are GTPases. In bacteria they are called EF-Tu and in eukaryotes EF-1. They contain 3 domains, one a GTPase, and 2 that bind the tRNAs. The ribosome functions as a GAP. If the tRNA has delivered the correct amino acid, then the fit between the tRNA and the mRNA will be proper, and this will lead to GTP hydrolysis by the EF-Tu, stimulated by the ribosome. The GDP-bound EF-Tu then needs a guanine nucleotide exchange factor, which is called EF-Ts.
If the anti-codons do not match the codons, then the ribosome will not stimulate GTP hydrolysis, and the EF-Tu-aa-tRNA complex will quickly dissociate as a unit. (Fig. 3-16)
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Copyright 2007, Steven B. Vik, Southern Methodist University