Structure of hammerhead ribozyme A ribozyme is an RNA molecule with a well defined tertiary structure that enables it to catalyze a chemical

reaction. Ribozyme means ribonucleic acid enzyme. It may also be called an RNA enzyme or catalytic RNA. Many natural ribozymes catalyze either the hydrolysis of one of their own phosphodiester bonds (self-cleaving ribozymes), or the hydrolysis of bonds in other RNAs. Some have been found to catalyze the aminotransferase activity of the ribosome. Examples of ribozymes include the hammerhead ribozyme, the VS ribozyme and the hairpin ribozyme. Investigators studying the origin of life have produced ribozymes in the laboratory that are capable of catalyzing their own synthesis under very specific conditions, such as an RNA polymerase ribozyme.[1] Mutagenesis and selection has been performed resulting in isolation of improved variants of the "Round-18" polymerase ribozyme from 2001. "B6.61" is able to add up to 20 nucleotides to a primer template in 24 hours, until it decomposes by hydrolysis of its phosphodiester bonds.[2] The "tC19Z" ribozyme can add up to 95 nucleotides with a fidelity of 0.0083 mutations/nucleotide.[3] Some ribozymes may play an important role as therapeutic agents, as enzymes which tailor defined RNA sequences, as biosensors, and for applications in functional genomics and gene discovery

Discovery

Schematic showing ribozyme cleavage of RNA. Before the discovery of ribozymes, enzymes, which are defined as catalytic proteins,[5] were the only known biological catalysts. In 1967, Carl Woese, Francis Crick, and Leslie Orgel were the first to suggest that RNA could act as a catalyst. This idea was based upon the discovery that RNA can form complex secondary structures.[6] The first ribozymes were discovered in the 1980s by Thomas R. Cech, who was studying RNA splicing in the ciliated protozoan Tetrahymena thermophila and Sidney Altman, who was working on the bacterial RNase P complex. These ribozymes were found in the intron of an RNA transcript, which removed itself from the transcript, as well as in the RNA component of the RNase P complex, which is involved in the maturation of pre-tRNAs. In 1989, Thomas R. Cech and Sidney Altman won the Nobel Prize in chemistry for their "discovery of catalytic properties of RNA."[7] The term ribozyme was first introduced by Kelly Kruger et al. in 1982 in a paper published in Cell.[8]

It had been a firmly established belief in biology that catalysis was reserved for proteins. In retrospect, catalytic RNA makes a lot of sense. This is based on the old question regarding the origin of life: Which comes first, enzymes that do the work of the cell or nucleic acids that carry the information required to produce the enzymes? Ribo-Nucleic acids as catalysts circumvents this problem. RNA, in essence can be both the chicken and the egg.[9] In the 1970s Thomas Cech, at the University of Colorado at Boulder, was studying the excision of introns in a ribosomal RNA gene in Tetrahymena thermophila. While trying to purify the enzyme responsible for splicing reaction, he found that intron could be spliced out in the absence of any added cell extract. As much as they tried, Cech and his colleagues could not identify any protein associated with the splicing reaction. After much work, Cech proposed that the intron sequence portion of the RNA could break and reform phosphodiester bonds. At about the same time, Sidney Altman, a professor at Yale University, was studying the way tRNA molecules are processed in the cell when he and his colleagues isolated an enzyme called RNase-P, which is responsible for conversion of a precursor tRNA into the active tRNA. Much to their surprise, they found that RNase-P contained RNA in addition to protein and that RNA was an essential component of the active enzyme. This was such a foreign idea that they had difficulty publishing their findings. The following year, Altman demonstrated that RNA can act as a catalyst by showing that the RNase-P RNA subunit could catalyze the cleavage of precursor tRNA into active tRNA in the absence of any protein component. Since Cech's and Altman's discovery, other investigators have discovered other examples of self-cleaving RNA or catalytic RNA molecules. Many ribozymes have either a hairpin or hammerhead shaped active center and a unique secondary structure that allows them to cleave other RNA molecules at specific sequences. It is now possible to make ribozymes that will specifically cleave any RNA molecule. These RNA catalysts may have pharmaceutical applications. For example, a ribozyme has been designed to cleave the RNA of HIV. If such a ribozyme was made by a cell, all incoming virus particles would have their RNA genome cleaved by the ribozyme, which would prevent infection.

[edit] Activity
Although most ribozymes are quite rare in the cell, their roles are sometimes essential to life. For example, the functional part of the ribosome, the molecular machine that translates RNA into proteins, is fundamentally a ribozyme, composed of RNA tertiary structural motifs that are often coordinated to metal ions such as Mg2+ as cofactors. There is no requirement for divalent cations in a five-nucleotide RNA that can catalyze transphenylalanation of a four-nucleotide substrate which has three base complementary sequence with the catalyst. The catalyst and substrate were devised by truncation of the C3 ribozyme.[10] RNA can also act as a hereditary molecule, which encouraged Walter Gilbert to propose that in the distant past, the cell used RNA as both the genetic material and the structural and catalytic molecule, rather than dividing these functions between DNA and protein as

they are today. This hypothesis became known as the "RNA world hypothesis" of the origin of life. If ribozymes were the first molecular machines used by early life, then today's remaining ribozymes -- such as the ribosome machinery -- could be considered living fossils of a life based primarily on nucleic acids. A recent test-tube study of prion folding suggests that an RNA may catalyze the pathological protein conformation in the manner of a chaperone Although most ribozymes are quite rare in the cell, their roles are sometimes essential to life. For example, the functional part of the ribosome, the molecular machine that translates RNA into proteins, is fundamentally a ribozyme, composed of RNA tertiary structural motifs that are often coordinated to metal ions such as Mg2+ as cofactors. There is no requirement for divalent cations in a five-nucleotide RNA that can catalyze transphenylalanation of a four-nucleotide substrate which has three base complementary sequence with the catalyst. The catalyst and substrate were devised by truncation of the C3 ribozyme.[10] RNA can also act as a hereditary molecule, which encouraged Walter Gilbert to propose that in the distant past, the cell used RNA as both the genetic material and the structural and catalytic molecule, rather than dividing these functions between DNA and protein as they are today. This hypothesis became known as the "RNA world hypothesis" of the origin of life. If ribozymes were the first molecular machines used by early life, then today's remaining ribozymes -- such as the ribosome machinery -- could be considered living fossils of a life based primarily on nucleic acids. A recent test-tube study of prion folding suggests that an RNA may catalyze the pathological protein conformation in the manner of a chaperone

Structural Biochemistry/Enzyme/Allosteric Enzymes

< Structural Biochemistry | Enzyme Jump to: navigation, search Allosteric enzymes are an exception to the Michaelis-Menten model. Because they have more than two subunits and active sites, they do not obey the Michaelis-Menten kinetics but instead have sigmoidal kinetics. An example of an allosteric enzyme is hemoglobin. Since allosteric enzymes are cooperative, a sigmoidal plot of V0 versus [S] results:

A sigmoidal plot has an S curve resulting from the combination of the T state and R state curves. The T state curve would be lower than the curve shown here, and the R state curve would be higher. Unlike many enzymes, allosteric enzymes do not obey MichaelisMenten kinetics. The reason for this is that allosteric enzymes must account for multiple active sites and multiple subunits. Thus, allosteric enzymes show the sigmodial curve shown above. The plot for reaction velocity, vo, versus the substrate concentration does not exhibit the hyperbolic plot predicted using the Michaelis-Menten equation. With allosteric enzymes, the catalytic activity affecting one substrate can alter the properties of other active sites located within the same enzyme. The result of this interaction equilibrium is a cooperative effect, meaning teh binding of the substrate to an enzyme's active site affects the binding of substrate to other active sites. This property of cooperativity accounts for the sigmodial curve of vo versus the concentration of substrate. A. Introduction Allosteric enzymes are unique compared to other enzymes because of its ability to adapt various conditions in the environment due to its special properties. The special property of Allosteric enzymes is that it contains an allosteric site on top of its active site which binds the substrate. The binding of a nonsubstrate molecule to the allosteric site functions to influences the activity of the enzyme. In influencing the activity, it can either enhance or impair the activity of the enzyme. Another important property of allosteric enzymes is

that it also contains many polypeptide chains with multiple active and allosteric sites. The nonsubstrate molecules that bind at the allosteric sites are called allosteric modulators. Example: A clear example of an allosteric enzyme is aspartate trascarbamoylase. The enzyme catalyzes the first step in the synthesis of pyrimidines. The enzyme functions to catalyze the condensation of aspartate and carbamoyl phosphate to form Ncarbamoylaspartate and orthophosphate. The enzyme ultimately catalyzes the reaction that will yield cytidine triphosphate (CTP). This allosteric enzyme is unique in that for high products of the final product CTP, the enzyme activity is low. However, for low concentrations of the final product CTP, the enzymatic activity is high. The allosteric nature is thus represented as the CTP molecule has a odd configuration or shape that is unlike the substrates. Rather than binding to the active site, CTP binds to the allosteric site. Thus, CTP functions as an allosteric inhibitor decreasing the enzymatic activity of the enzyme. This enzyme also has separate regulatory and catalytic subunits on separate polypeptide chains. There are instances though when CTP concentrations remain high and cells in the body need more enzyme. This is when a different allosteric molecule ATP functions to attach to the allosteric site and functions as enzyme activator enhancing the activity of the enzyme. Thus, even with high concentrations of CTP, the enzyme activity could be enhanced because of ATP, which also acts on the allosteric site. This example explains the benefits of allosteric control and the ability allosteric enzymes to adapt to various conditions of the environment. This is particularly helpful for cells because there are occasions when the cell requires an allosteric activator like "ATP" to enhance the enzyme even when it is inhibited due to high amounts of product. (CTP) The aspect of feedback inhibition is represented as well as high amounts of product acts to inhibit the action of the enzyme acting in a inhibitory manner. B. Properties of Allosteric Enzymes There are distinct properties of Allosteric Enzymes that makes it different compared to other enzymes. (1) One is that allosteric enzymes do not follow the Michaelis-Menten Kinetics. This is because allosteric enzymes have multiple active sites. These multiple active sites exhibit the property of cooperativity, where the binding of one active site affects the affinity of other active sites on the enzyme. As mentioned earlier, it is these other affected active sites that result in a sigmoidal curve for allosteric enzymes. (2) Allosteric Enzymes are influenced by substrate concentration. For example, at high concentrations of substrate, more enzymes are found in the R state. The T state is favorite when there is an insufficient amount of substrate to bind to the enzyme. In other words, the T and R state equilibrium depends on the concentration of the substrate. (3) Allosteric Enzymes are regulated by other molecules. This is seen when the molecules 2,3-BPG, pH, and CO2 modulates the binding affinity of hemoglobin to oxygen. 2,3-

BPG reduces binding affinity of O2 to hemoglobin by stabilizing the T- state. Lowering the pH from physiological pH=7.4 to 7.2 (pH in the muscles and tissues)favors the release of O2. Hemoglobin is more likely to release oxygen in CO2 rich areas in the body.

Mechanism of Enzyme Action

Introduction - Enzyme Characteristics: The basic mechanism by which enzymes catalyze chemical reactions begins with the binding of the substrate (or substrates) to the active site on the enzyme. The active site is the specific region of the enzyme which combines with the substrate. The binding of the substrate to the enzyme causes changes in the distribution of electrons in the chemical bonds of the substrate and ultimately causes the reactions that lead to the formation of products. The products are released from the enzyme surface to regenerate the enzyme for another reaction cycle. The active site has a unique geometric shape that is complementary to the geometric shape of a substrate molecule, similar to the fit of puzzle pieces. This means that enzymes specifically react with only one or a very few similar compounds. Lock and Key Theory: The specific action of an enzyme with a single substrate can be explained using a Lock and Key analogy first postulated in 1894 by Emil Fischer. In this analogy, the lock is the enzyme and the key is the substrate. Only the correctly sized key (substrate) fits into the key hole (active site) of the lock (enzyme). Smaller keys, larger keys, or incorrectly positioned teeth on keys (incorrectly shaped or sized substrate molecules) do not fit into the lock (enzyme). Only the correctly shaped key opens a particular lock. This is illustrated in graphic on the left. QUES: Using a diagram and in your own words, describe the various lock and key theory of enzyme action in relation to a correct and incorrect substrate.

Induced Fit Theory: Not all experimental evidence can be adequately explained by using the so-called rigid enzyme model assumed by the lock and key theory. For this reason, a modification called the induced-fit theory has been proposed. The induced-fit theory assumes that the substrate plays a role in determining the final shape of the enzyme and that the enzyme is partially flexible. This explains why certain compounds can bind to the enzyme but do not react because the enzyme has been distorted too much. Other molecules may be too small to induce the proper alignment and therefore cannot react. Only the proper substrate is capable of inducing the proper alignment of the active site. In the graphic on the left, the substrate is represented by the magenta molecule, the enzyme protein is represented by the green and cyan colors. The cyan colored protein is used to more sharply define the active site. The protein chains are flexible and fit around the substrate. Carboxypeptidase - Chime in new window Nature of Active Site and Substrate Interaction: Enzymes have varying degrees of specificity. Some enzymes have absolute specificity for one substrate and no others, while other enzymes react with substrates with similar functional groups, side chains, or positions on a chain. The least specific enzymes catalyze a reaction at a particular chemical bond regardless of other structural features. Much experimental work is devoted to gaining an understanding of the nature of the active site in an enzyme. Since enzymes are proteins, the nature of amino acid side chains in the vicinity of the active site is important. The specific amino acid side chains have been determined for many enzymes. The active site for carboxypeptidase A will be used to illustrate the principles involved as shown in the graphic on the left. The substrate (space filling gray,blue red) can interact with the active site through opposite charges, hydrogen bonding (shown in yellow), hydrophobic non-polar interaction, and coordinate covalent bonding to the metal ion activator as shown in magenta. The numbers behind the amino acids indicate the sequence position of the amino acid in the protein. The white lines represent the wire frames of the other amino acids in the enzyme. The carbonyl bond is activated by interaction with the Zn ions. This leads to the addition of -OH from water to the carbonyl to produce an acid and the ultimate rupture of the C-N bond. Carboxypeptidase - Chime in new window