Enzymes cont.

Uncompetitive Inhibition In this type of inhibition the inhibitor binds to a site that is distinct from the substrate binding site. They bind ONLY to the ES complex (forming ESI complex) and do not resemble the substrate. As such this inhibitor does not affect the efficiency of substrate binding, only the catalytic function of the enzyme itself. Mixed Inhibition The inhibitor binds to either the enzyme or the enzyme-substrate complex. As such this can affect both substrate binding efficiency, as well as catalytic efficiency. Chymotrypsin Chymotrypsin is a protease that hydrolyses peptide bonds. Chymotrypsin is secreted by the pancreatic cells in the duodenum. This enzyme recognizes and cleaves peptide bonds that are adjacent to bulky hydrophobic amino acids such as: Phe, Tyr, Trp. Chymotrypsin starts off as an inactive precursor known as chymotrypsinogen. Chymotrypsinogen is 245 amino acids in length and composed of multiple segments held together by disulfide bonds. Trypsin proteolytically cleaves chymotrypsinogen between Arg15-Ile16 which forms the activated chymotrypsin. This -chymotrypsin then proceeds to cut out Ser14-Arg15 and finally Thr147-Asn148 forming the completely active enzyme. Three chains of chymotrypsin are linked by two disulfide bonds. Within chymotrypsins catalytic site, His57,Arg102 and Set195 are contained and brought very close together for interaction. The catalytic process of chymotrypsin involves the formation of an acyl enzyme intermediate. A peptide bond is cleaved and an ester linkage is formed between the peptide carbonyl carbon and the enzyme. Deacylation occurs via a hydrolysis reaction of the ES ester bond resulting int he release of the modified substrate and regeneration of the enzyme. The study of chymotrypsin indicated that it had a rapid burst phase followed by a slower steady state phase independent of substrate concentration. In the specific study of chymotrypsin's action on pNitrophenylacetate, it was seen that in the burst phase p-Nitrophenolate was rapidly released and there was the formation of a acyl enzyme intermediate and acetate remains linked to the enzyme. The acyl enzyme intermediate is then slowly hydrolyzed to release acetate ion. The burst phase of this catalytic process corresponds to just one molecule of pnitrophenol being released for every molecule of enzyme present. Important amino acids - Ser195 the enzymes active Ser residue -His57 essential active site residue -Asp195 Together these amino acids form a catalytic triad in a hydrogen bonding network. Binding of a peptide substrates causes a conformational change in chymotrypsin, compressing the H-bond between His57 and Asp102 resulting in an even stronger interaction between the two residues. This increases the pKa of histidine from 7 to >12, causing His57 to act as a base which can remove a proton form Ser195 making it a strong nucleophile. Later during the series of reactions, His57 can act as a proton donor. The rate determining step of this catalytic process is when Ser195 attacks the peptide carbonyl forming a tetrahedral intermediate

Mode of action - Chymotrypsin

1. Binding of the substrate to the enzyme forming Michaels Complex 2. Rate determining step- Ser195 attacks peptide carbonyl forming a tetrahedral intermediate complex 3. Imidazole of His57 activates Ser195 - Takes H+ from Ser195's OH 4. Asp102 polarizes His57 but doesn't remove a proton from the imidazole ring 5. Tetrahedral intermediate decomposes the acyl-enzyme intermediate through the donation of N3 of His57 RNH2, is released to be replaced by water 6. Acyl-Enzyme intermediate is deacylated by attack of water-oxygen on the carbonyl carbon 7. His57 picks up another H+ from water forming a second tetrahedral intermediate 8. Carboxylate product is released and the enzyme is regenerated, water is the nucleophile and Ser195 is the leaving group

Nucleotides and Nucleic Acids The Basics

- Used as a source of energy ATP,GTP - Deoxyrbonucleic acid or ribonucleic acid - cellular genetic information contained within NA bases of DNA and mRNA - nucleic acid sequence determines the sequence of proteins - NA base sequence of mRNA coded by nucleotide sequence of DNA - Genes are the segments of DNA that code for proteins or RNA Two Types of Nucleic Acid Bases

Purines - Adenine and Guanine Pyrimidines - Cytosine, Thymine and Uracil Both of these are planar aromatic heterocyclic molecules Nucleotides have a phosphate group, which forms a phosphate ester on their 5' carbon. There are two distinct ribose derivatives, one of which has a OH consituent on the 2' and 3'C. The other ribose derivative only has an OH group on the 3'C, this is known as a deoxynucleotide. Purines and pyrimidines also differ in the ways in which they are attached to ribose rings. Pyrimidines are attached to the 1'C of the ribose ring through it N1. Purines on the other hand are attached to the 1'C of the ribose ring through its N9. Nucleotides in both DNA and RNA are linked to one another through phosphate group bridges (phosphodiester bonds) in which the 5'phosphate group is linked to the 3'hydroxyl group of the next nucleotide. The backbone of these nucleic acids, made up of multiple nucleotides, is composed of alternating phosphate-ribose residues. The nucleic acid bases are then the side groups, joined to the backbone at regular intervals. The nucleic acid backbone is hydrophobic. The phosphate groups linking the nucleotides have a pka = 0 so will by unprotonated at physiological pH. Free pyrimidines and purines are however weakly basic, hence why they are called bases. Short series of nucleic acids linked together are known as oligonucleotides. The 5' end of the oligonucleotide contains a free phosphate group, and the last nucleic acid on the 3' end has a free hydroxyl group.

History of DNA
- First isolated by Friedrich Miescher in 1868 - up until the '40s it was thought proteins contained genetic info - Avery,Macloead, McCarty: DNA was transforming material in Strep - Hershey and Chase: Radiolabelled DNA from virus infected bacteria Chargaffs Rules: - base composition or DNA varies between species - DNA from different tissues in individual species has same base composition - base composition will not change due to age or environment - A=T, G=C Watson and Crick: - obtain x-ray diffraction data from Franklon - diffraction pattern showed two

ENZYMES
Characterized by formation of the Enzyme- Substrate Complex E +S ES Binding of the substrate occurs within a pocket of the enzyme known as the active site Enzymes speed up reactions by lowering the activation energy ( G) enhancing the reaction rate The energy used to speed up reaction rates comes mainly from weak interactions between enzyme and substrate, which occur during the transition state and act to stabilize it Understanding an enzyme and its action fully includes: - Studying the 3D structure of the enzyme - Using mutagenesis studies to determine roles of specific amino acids in eznymes - determining the kinetic parameters of the processes occurring during the reaction The concentration of substrate [S] is a key affecter of enzyme reaction rates, varying [S] will change the overall Velocity of a reaction. The [S] of most reactions is quite constant within the initial period of reactions. It may be valid to measure the Vo of a reaction which corresponds to the slope of the initial curve of the reaction.

Enzymes Cont.
The concentration of free [ES] and [E] cannot be measured by themselves and thus the total enzyme concentration [E]t must be measured [E]t = [E]+[ES]. [ES] cannot be measure by itself, however it can be calculated using the equation [ES] = [E]t * [S] / (Km + [S]) where Km is the Michaels Constant. The initial velocity of a reaction can be calculated using the equation Vo = k2[E]t[S]/(Km + [S]) Measuring the initial velocity before more than 10% of the substrate has been converted into product minimizes complication factors due to the effects of: - reversible reactions -inhibition of enzymes by product formation - progressive inactivation of enzymes The maximum velocity of a reaction occurs when at substrate concentrations which completely saturate the enzymes aka when all enzymes are in enzymesubstrate complexes. Vmax = k2[E]t ,allowing us to derive the equation: Vo = Vmax[S]/(Km + [S]) Michaelis Menton Equation This occurs when Vo is 1/2 Vmax. The Michaels constant Km varies according to the enzyme, and is also a function of temperature and pH. Km = Ks + (k2/k1) where Ks is the dissociation of the Michaels complex . Km could also be used to measure the enzyme affinity for the substrate, this of course is assuming that k2/k1 is small compared to ks . The Lineweaver-Burk (double reciprocal plot) is widely used to determine important terms in enzyme kinetics, such as Km and Vmax. It is the reciprocal of the MM equation: 1/ V0 = (KM /Vmax ) (1/[S]) + 1/ Vmax The slope of this plot is Km/Vmax , the intercept with the line 1/vo gives you 1/Vmax and you can use the extrapolated value of 1/[S] to give you 1/Km . The disadvantages in the use of the Line-Weaver Burk plot include: - experiments using this method must involve large values of [S] - Small values of [S] produce small errors in Vo but larger errors within 1/V0 producing errors in Km and Vmax

For most reactions the Vo will increase in a linear fashion with the increase of [S], but will eventually begin to plateau as the concentration of [S] gets higher. This plateau is known as the Vmax of a reaction.

Most enzymatic reactions occur as a two stage process which can be described by E+S ES P+E . The reactions rate limiting step occurs when the [S] is high enough to convert all the enzyme to ES, so the second step is rate limiting. The rate of reaction V is described as V = d[P]/dt = k2[ES] where d[P]/dt is the rate of Product production. The overall rate of production of the enzyme-substrate complex also known as the Michaels complex, is the difference between the rate of the ES production and the rate of its decomposition, described by Ks = [E][S]/[ES] = k-1/k1. This allows us to derive the equation d[ES]/dt = k1[E][S] - k-1[ES] - k2[ES] With the exception of the very early phase and the end phase of the reaction free enzyme [E] and [ES] is constant as long as [S] > [E]. AKA the formation of ES = E +S, a steady state is maintained.

An enzymes catalytic constant (kcat) is a measure of its catalytic efficiency and can be derived from the equation Kcat = Vmax/[E]t is a measure of the reactions maximum velocity and its true enzyme concentration. Enzymatic inhibitors Two Major Classes of Inhibitors: - Reversible - Irreversible Competitive Inhibition Occurs when there is competition between the substrate and the inhibitor for the active site of the enzyme. The inhibitor is close enough in form to the substrate that the inhibitor will bind to the enzyme to form ES-like complex. This EI is short lived but will still interfere with the enzyme efficiency. Structure and shape of inhibitor offer insights into how the normal substrate binds