Lecture 28 March 2005

Catalysts accelerate the rate of a reaction without undergoing any permanent change.

Most biological catalysts are proteins, and are called enzymes. RNA molecules can also be catalysts, such as the large subunit of the ribosome, and are termed ribozymes.

The catalysis of chemical reactions by enzymes can be extremely rapid, if one compares those rates to uncatalyzed rates. The decarboxylation of orotidine 5'-monophosphate to uridine 5'-monophosphate is extremely slow, but is enhanced by 17 orders of magnitude by the enzyme orotidine 5'-monophosphate decarboxylase (ODCase). (Fig. 2-19)

Other enzymes only accelerate reactions by a few orders of magnitude, shown Fig. 2-20.

Remember that chemical reactions, or transformations, can be thought of in terms of an equilibrium. The associated equilibrium constant can be described by the concentration of reactants and products at equilibrium, or by the ratio of the forward and reverse rate constants. A catalyst does not affect the concentrations at equilibrium. It does increase the forward rate constant, but it affects the reverse rate by the same factor. And so, the ratio (the equilibrium constant) is not changed. By increasing the forward rate constant, a catalyst increases the rate of product formation, and shortens the time to reach equilibrium.

Enzymes generally use multiple different ways to catalyze reactions. There is not a single explanation for the power of enzymatic catalysis. We will explore some of the different means of catalysis. They include proximity and orientation of substrates with catalytic groups of the enzyme. Microenvironments at the binding sites can also enhance the catalytic properties of amino acid side chains and of co-factors.

Transition state theory is often used to analyze enzyme-catalyzed reactions. The reaction proceeds through a high energy state, or barrier, that is called the transition state. The energy required to overcome this barrier is the activation energy. The transition state is the structure of the highest free energy. Catalysis requires that the free energy barrier be reduced. This can be accomplished by simply stabilizing the transition state, or the enzyme can cause the reaction to take a different path, perhaps with several intermediates along the way, resulting in several lower barriers. (Fig. 2-21)

Active-Site Geometry

In general, substrates must find the active-site of an enzyme by random collisions. For charged substrates, there can be an attractive potential around the enzyme, that allows the substrate to be pulled towards the active-site. An example is Cu-Zn superoxide dismutase (Fig. 2-22).

Electrostatic interactions can also help to orient the substrate in the active-site (Fig. 2-23)

Once at the active-site, a variety of groups from the enzyme will help to orient the substrate precisely, such that the catalytic groups are all properly placed. Co-factors may be involved in the catalysis also. (Fig. 2-24).

Sometimes the groups of the enzyme that interact with the substrate can be divided into those that contribute to specificity and those that contribute to reactivity. In that case, it might be possible to change the specificity of the enzyme without affecting its reactivity, through site-directed mutagenesis.

The Proximity Factor

Some enzymes promote catalysis primarily through simple proximity effects. In other words, it may be sufficient to simply bring the two substrates together, in the proper orientation, and at a close distance, to cause a reaction. This is called the proximity factor. An example is aspartate transcarbamoylase (ATCase), which is an important enzyme in pyrimidine biosynthesis.

carbamoyl-phosphate + aspartate → N-carbamoyl-aspartate + phosphate

A co-crystal of the enzyme with the inhibitor PALA (N-phosphoacetyl-L-aspartate) shows that no catalytic groups appear to be near the substrates. (Fig. 2-25). The reactivity comes from the substrates themselves, as they are held close together.

Ground-State Destabilization

The enzyme chorismate mutase appears to destabilize its substrate chhorismate, by forcing it into an unusual "chair" conformation. This promotes catalysis because the conformation is on the path to the product. That is, it resembles the transition-state. (Fig. 2-26).

Stabilization of Transition States

Enzymes are often complementary to the transition state of the reaction they catalyze. That includes both shape complementarity and charge complementarity. This reduces the free energy of the transition state, or lowers the barrier. (Fig. 2-27) The enzyme might also stabilize the substrate, i.e. bind it tightly. But, in order to be a catalyst, the enzyme must stabilize the transition state more than it does the substrate.

In the example of citrate synthase, the enzyme stabilizes the charge distribution that must develop during the reaction:

oxaloacetate + acetyCoA → citrate + CoA

Two carbonyl groups must become partially negative, and these are stabilized by positively-charged Histidine residues at the active site of citrate synthase (Fig. 2-28)

The complementarity necessary for transition state stabilization might require a conformational change in the enzyme, such as in Phosphoglycerate kinase (Fig. 2-29) You can see the change in secondary structure of a loop on the left, and the binding of an essential Magnesium ion (green).

Exclusion of Water

Some enzyme use the exclusion of water from the active site to improve catalysis. This is true for enzymes that transfer Hydride ions (H^-) from NADH. This is important since water would tend to prevent such a transfer. It is accomplished by having a loop of the protein fold over the substrates after they are bound. (Fig. 2-30) (Jmol) The example shown is lactate dehydrogenase.

lactate + NAD → pyruvate + NADH

Although the loops must open and close each time the enzyme turns over, the actual rate of motion is not fast since the distances (10 Å) are so small.

Redox Reactions

First of the four common types of reactions (Fig. 2-31)

They include oxidation of Carbons, from CH to COH, or COH to C=O, and desaturation of C-C bonds.

They typically require cofactors such as NADH, NADPH, flavins FAD or FMN, or metals.

Addition/Elimination Reactions

Second of the four common types of reactions

Examples include the addition of water to the double bond of fumarate (Fumarase), and the addition of acetate to the carbonyl carbon of oxaloacetate (Citrate synthase). (Fig. 2-32)

Hydrolysis Reactions

Third of the common types of reactions

Examples include hydrolysis of proteins (Trypsin) and nucleic acids (Ribonuclease). These are favorable reactions, and they can be reversed to make proteins or nucleic acids, if the substrates are activated first through phospho-esters. (Fig. 2-33)

Decarboxylation Reactions

Fourth of the common types of reactions.

The removal of a Carbon atom is difficult, so to shorten a carbon chain, it usually proceeds through a decarboxylation. So, the last Carbon is oxidized to a carboxyl group, and then removed. Examples include pyruvate decarboxylase, which converts pyruvate to acetaldehyde + carbon dioxide. The stability of carbon dioxide makes these reactions more favorable. (Fig. 2-34)

Active-Site Chemistry: Acid-base chemistry

The transfer of a proton from the substrate to the enzyme, or from the enzyme to the substrate, is the most common element of enzyme catalysis.

Protons are transfered from groups of low affinity (acids) to groups of high affinity (bases). The affinity is measured by the pK_a value, which is the pH at which 50% of a group is protonated.

Strong acids have pK_a values less than 2, while strong bases have pK_a values greater than 12. Strong acids are essentially totally dissociated at pH 7, while strong bases are toatlly protonated at pH 7.

Weak acids have pK_a values between 4 and 7, while weak bases have pK_a values between 7 and 10. They are all partially dissociated at pH 7. Examples are shown in Fig. 2-35.

In protein environments the pK_a values can be shifted by several units from the standard values. A carboxyl group, such as the side chain of glutamate, can have its pK_a value shifted up (making it a weaker acid) by a nonpolar environment. The charged form (negative here) is destabilized by a nonpolar environment, relative to an aqueous environment. That shifts the pK_a up, since it will take a higher pH to deprotonate the group. This occurs in the enzyme lysozyme, which utilizes a protonated glutamic acid at its active site. (Fig. 2-36)

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, bu 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

Final topic: Some enzymes have domains with non-enzyme functions

These functions include roles in regulation of transcription or translation, or signalling pathways.

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