A2 LEVEL. A chain COOH. Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn H 2 N. B chain

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1 Blood insulin concentration Meal atural insulin release in nondiabetic person Diabetic person after insulin injection Insulin injection Time/h Figure 7 Insulin concentrations in the blood of a diabetic person following an insulin injection (injections also contain a slowacting form of insulin which produces an effect for up to 12 hours; after that the insulin level falls to zero). Insulin injection Blood kin and muscle tissue Muscle tissue Blood capillary Figure 9 Injecting insulin. lthough the insulin hexamers are too large to pass through the blood capillary membrane, the monomers are able to do so. chain 2 2 ly Ile Val lu ln ys ys Thr er Ile ys er Leu Tyr ln Leu lu sn Tyr ys sn 1 B chain 5 10 he Val sn ln is Leu ys ly er is Leu Val lu la Leu Tyr Leu Val ys ly lu rg ly 15 Thr Lys ro Thr 30 he Try he 25 Figure 11 uman insulin. The two chains are held together by links.

2 ribose base: uracil (U) phosphate 2 base: cytosine () 2 base: adenine () 2 base: guanine () sugarphosphate backbone bases 2 U 2 2 sugar phosphate backbone or simply U bases U (a) (b) (c) Figure 14 epresentations of the structure of : (a) how groups join together; (b) a skeletal formula; and (c) two simpler ways of showing the structure.

3 group forms an ester link to the amino acid in this case alanine (la) (a) U U Messenger (m) carries the code for protein synthesis m (b) t la la nticodon for binding to a codon on m: in this example it would bind to, the codon for alanine t t codon m Figure 15 chematic representation of a t molecule showing the three bases which form the anticodon. (c) ibosome Transfer (t) collects an amino acid and takes it to the m strand ibosomes contain ribosomal (r) They move along the m chain, reading the code and catalysing protein synthesis Figure 16 The roles of the different types of. 3 aving delivered its amino acid, t leaves the ribosome 2 mino acids are assembled into the growing protein chain is Leu t Figure 17 rotein synthesis and the reading of codons on m. U Val t ibosome lu t U U 2 2 U ibosome moves along the m chain ly Ile Val lu ln ys ys Thr er Ile ys er Leu Tyr ln Leu lu sn Tyr ys sn he Val sn ln is Leu ys ly er is Leu Val lu la Leu Tyr Leu Val ys ly lu rg This part of the insulin chain is being assembled below t la m ly he Thr Lys ro Thr Try he 1 Transfer (t) molecules bring amino acids to the m in the ribosome

4 chain or U chain hydrogen bonding between uracil and adenine chain hydrogen bonding between cytosine and guanine T T T T ugar phosphate backbone Figure 20 n illustration of the D double helix. or chain Figure 18 Molecular recognition and bases on (the symbol is used to represent two hydrogen bonds; represents three hydrogen bonds). D ell nucleus Membrane of nucleus m carries the codons for a protein from the nucleus to the ribosomes where protein synthesis takes place m t collects amino acids r in ribosomes ell material outside nucleus Figure 23 summary of protein synthesis in higher organisms. rotein

5 ome bacterial cells, unlike human cells, contain plasmids tiny circular pieces of D which are able to pass between cells plasmid ther bacterial D (in reality this is much bigger than the plasmid) The human gene responsible for insulin production can be built up from insulin m using viruses uman gene The modified plasmid is put back into bacterial cells ther enzymes reform sugar phosphate links and splice the human gene into the plasmid lasmids can be extracted and cut with restriction enzymes which break a sugar phosphate link in the D backbone The cells multiply in the fermenter The modified bacteria produce human insulin ( ) from the insulin gene Waste bacterial cells are destroyed The protein is extracted and modified if necessary to give the final product Figure 29 n illustration of the general approach used to produce a sample of insulin by genetic engineering. (a) (b) Figure 34 The secondary structure of a protein involves folding as a result of hydrogen bonding. This figure shows the protein chain folded into (a) a helix and (b) a sheet.

6 The centre of the hexamer contains polar groups which coordinate with Zn 2+ ion onpolar regions are brought into contact by dimerisation emaining nonpolar regions are brought into contact in the hexamer The outside of the hexamer is almost totally polar and interacts strongly with water Figure 38 n insulin hexamer. Insulin monomers Figure 40 ribbon diagram of the insulin hexamer. B9 Insulin B chain Leu ys ly er is Leu mino acids 6 11 Insulin gene in D TT TT T T TT trand 1 codons for insulin B chain trand 2 complementary D strand T eparation of double helix of D trand 1 trand 2 Figure 45 art of the human insulin gene which codes for the B chain around B9 (serine). TT TT T TT

7 1 ynthetic D (shown in green) sticks to correct sequence of bases on trand 2 trand 2 TT TT T T TT 2 ormal cell processes recreate the plasmid double helix TT trand 1 trand 2 TT T T TT Figure 46 The cell recognises the small piece of synthetic D and incorporates it into a plasmid. Figure 47 The cell tolerates a change to one of the bases in the piece of D and incorporates the synthetic D into a plasmid. When the cell divides, two different plasmids are formed one carries the normal human insulin gene, the other the gene for modified insulin. Blood insulin concentration Meal Diabetic person after modified insulin injection Diabetic person after human insulin injection Insulin injections Time/h Figure 48 Insulin concentrations in diabetic patients using human insulin and modified insulin: human insulin is injected 30 min before the meal; modified insulin is injected immediately before the meal. trand 2 trand 1 carrying 'mistake' T codon for sp TT TT T T T TT egion where bases on different strands cannot interact riginal plasmid carrying codon for er at B9 ew type of plasmid carrying codon for sp at B9 TT TT T T TT TT TT T T TT ubstrate binds reversibly eaction is catalysed roducts leave Enzyme ubstrate Enzyme substrate Enzyme product Enzyme roducts complex complex E + E E E + Figure 52 Illustration of the lock and key model of enzyme catalysis.

8 Without enzyme With enzyme ctivation enthalpy for uncatalysed reaction Enthalpy eactants ctivation enthalpy for enzymecatalysed reaction rogress of reaction roducts E + E E E + Figure 53 Lowering of the activation enthalpy barrier in an enzymecatalysed reaction.