enzymes 3 + 4

how large is the active site?

10-15 residues, only 2-3 directly participate in catalysis

what 2 things dos active site influence the most

catalytic activity and substrate specificity

what does the rest of the enzyme do apart from active site?

maintains the correct shape of active site

what are the 3 models of substrate binding

lock and key, Fischer 1894

flexible, Pauling 1946

induced fit, Koshland 1958

what is the problem with the lock and key model

does not explain transition states or why conformational changes occur in experiments

example of low specificity enzyme

papain, cleaves any peptide bond

example of high specificity enzyme

thrombin, cleaves only Arg-Gly bond within a specific sequence

what is binding energy

the free energy released when a substrate binds to the enzyme via multiple weak interactions, this stabalises TS, lowers AE

what type of bonds release binding energy

h bonds, ionic, Van der Waals

what is an important concept of binding energy

enzymes are evolved to bind to the TS more tightly than substrate, if only enzyme substrate complex is stabilised, AE increases and rate of reaction decreases

example of serine protease

chymotrypsin

how is chymotrypsin activated

first produced as inactive pancreas form chymotrypsinogen, tyrpsin then cleaves between Arg-15 and Ile-16 forming partially active pi-chymotrypsin, further cleaves itself to alpha-chymotrypsin, now fully active

what are serine proteases

enzyme family that catalyses the hydrolysis of peptide bonds, have highly reactive serine in active site

3 regions of active site in chymotrypsin

1. activated site

2. oxyanion hole

3. large hydrophobic pocket

what R groups does endopeptidase chymotrypsin cleave?

aromatic and bulky, Phe, Tyr, Trp

what R groups does endopeptidase trypsin cleave?

+charged, Arg, Lys

what R groups does endopeptidase elastase cleave?

small, hydrophobic, Ala

mechanism of chymotrypsin

serine positioned for deprotonation

upon substrate binding:

• His removes Ser H, Ser becomes nucleophilic

• Ser attacks carbonyl of peptide forming oxyanion teterahederal intermediate

• covalent acyl-enzyme intermediate is formed

hydrolysis releases first product

water attacks acyl-enzyme and second product is released

what is the function of restriction endonucleases e.g EcoRI

cleaves DNA at cognate sequences, requires Mg2+ as a cofactor - don’t form covalent intermediates unlike serine proteases

what is the mechanism of restriction endonucleases e.g EcoRI

metal io stabilises developing negative charge during single step hydrolysis

what is the scanning mechanism of restriction endonucleases e.g EcoRI

enzyme moves along DNA scanning for cognate sequences, in absence of Mg2+ enzymes bind incorrect and correct sequences, Mg2+ is essential for discrimination and activity

what are the 5 main enzyme regulation strategies

control of enzyme quantity

isoenzymes

allosteric control

proteolytic activation

covalent modifications

what is the mechanism of control of enzyme quantity?

regulates expression using TFs, transcription and translation

regulates proteolysis by tagging for degradation and directing to proteosome

what is the speed of control of enzyme quantity?

very slow, hours to days, allowing adaptation to long term environmental changes

evaluate control of enzyme quantity for regulation

poor responsiveness to rapid environmental change, irreversible

example of control of enzyme quantity

liver enzymes involved in gluconeogenesis are upregulated during fasting

what is proteolytic activation

enzyme is synthesised in inactive form, and activated by irreversible cleavage of specific bonds

evaluate proteolytic activation in enzyme regulation

+ fast

- irreversible

example of proteolytic activation

chymotrypsinogen → chymotrypsin, uses trypsin to cleave at Arg15-Ile16, forming pi and then alpha chymotrypsin

blood clotting fibrinogen → fibroin using thrombin

what is allosteric control

regulation by binding of small molecules to sites distinct from active site, conformation increases or decreases activity, often forming feedback loops

what re feedback loops?

a product produced late in a pathway that inhibits the enzyme that acts earlier in the reaction - allosteric control

example of allosteric control

aspartate transcarbamoylase catalyses 1st step in pyridine biosynthesis, inhibited by CTP via feedback inhibition

evaluate allosteric control

+ rapid ad reversible

what is covalent modification?

reversible addition/removal of functional groups via covalent bonds, most common is phosphorylation of -OH residues on serine, threonine and tyrosine

evaluate covalent modification

+ reversible

+ fast

- requires ATP

example of covalent modification

Cdks control cell cycle, activated only when:

• phosphorylated by a kinase

• dephosphorylated on seperate site

• bound to cyclin protein