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Lecture 15 Use of protease specificity in molecular biology
Biomolecules
University of Lincoln
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The mechanism of chymotrypsin ● Chymotrypsin is a protease
● The determination of the three-dimensional structure of chymotrypsin by David Blow in 1967 as a source of further insight into its mechanism of action ● Overall, chymotrpsin is roughly spherical and comprises three polypeotide chains, linked by disulfide bonds ● It is synthesised as a single polypeptide, termed chymotrypsinogen, which is activated by the proteolytic cleavage of the polypeptide to yirld the three chains
● The hydrophobic pocket of chymotrypsin is responsible for its substrate specificity ● The key amino acids that constitute the binding site are labelled, including the active-site serine residue (boxed) ● The position of an aromatic ring bound in the pocet is shown in green ● The active site of chymotrypsin, marked by serine 195, lies in a cleft on the surface of the enzyme
● The active site of chymotrypsin, marked by serine 195, lies in a cleft on the surface of the enzyme. The structural analysis revealed the chemical basis of the special reactivity of serine 195 ● The side chain of serine 195 is hydrogen bonded to the imidazole ring of histidine 57 ● The NH group of this imidazole ring is hydrogen bonded to the carboxylate group of aspartate 102 ● This constellation of residues is referred to as the catalytic triad
● After substrate binding (step 1), the reaction begins with the hydroxyl group of serine 195 making a nucleophillic attack on the carbonyl carbon atom of the substrate (step 2)
● The ester group of the acyl-enzyme is now hydrolysed by a process that is essential a repeat of step 2, 3 and 4 ● The water molecules attacks the carbonyl group while a proton is concomitantly removed by the histidine residue, which now acts as a general acid catalyst, forming a tetrahedral intermediate (step 6) ● This structure breaks down to form the carboxylic acid product (step 7) ● Finally, the release of the carboxylic acid product (step 8) readies the enzyme for another round of catalysis
Use of protease specificity in molecular biology
● The peptide bond nearest to the amino terminus of the protein or polypeptide is cleaved in two steps ● The two steps are carried out under very different reaction conditions ○ Basic conditions in step 1 ○ Acidic conditions in step 2 ● Allowing one step to proceed to completion before the second is initiated
● Protein sequencing (fragmentation) ○ Peptides longer than about 50-70 amino acids long cannot be sequenced reliably by the Edman degradation ○ Because of this, long protein chains need to be broken up into smaller fragments and purified so they cna then be sequenced individually ○ Digestion is done either by endopeptidases such as trypsin or pepsin or by chemical reagents such as cyanogen bromide and NTCB (2-nitro-5- thiocyanobezoic acid) fragmentation ○ Different enymes give different cleavage patterns, and the overlap between fragments can be used to construct an overall sequence
classify the following amino acids as nonpolar, polar but uncharged, positively charged or negatively charged: ● leucine - non polar ● aspartate - negatively charged ● lysine - positively charged ● glutamine - polar but uncharged
a helices have a repeating pattern of hydrogen bonding. What is this pattern? ● CO of residue i to NH of residue i+
how does the hydrogen bonding found with B sheets differ from those found within a helices? ● hydrogen bonds are formed between atoms within adjacent strands of a B sheet, not the same strand as in a helices
histidine is often present within enzyme active sites. explain why. ● it is positively charged or neutral around pH 7 which allows it to donate a proton or
accept a proton
a glutamate is at position one of an alpha helix (below) whilst an arginine is at position four. why are the relative positions and chemical properties of these residues important with respect to noncovalent bonding within this alpha helix? ● the position within the helix place the two R groups close together ● glutamic acid and arginine carry charges of opposite polarity within their R groups allowing the formation of hydrogen bonding and electrostatic interactions - also known as a salt bridge
if positions 2, 6, 9 and 10 were occupied by isoleucine, valine, alanine and methionine, then what would you conclude about the properties of this helix? ● this face of the helix is hydrophobic
write the one letter codes for these amino acids in the same order as the amino acids are written above ● I, V, A, M
Lecture 15 Use of protease specificity in molecular biology
Module: Biomolecules
University: University of Lincoln
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