Molecular Machinery

• Molecular basis of cell function– Structure vs. Function– Molecular mechanisms

• ENZYME ACTION• Na+ K+ pump• Cell Signalling

ENZYMES• Hydrolase hydrolysis• Phosphatase REMOVE phosphate gps• Protease break down proteins• Nuclease break down nucleic acids• ATPase hydrolyse ATP

• Kinase ADD phosphate gps• Synthase join molecules• Polymerase join molecules (to make

polymers)

• Oxidoreductases electron transfer• Isomerases isomerise p49-50 text

How do Enzymes work?

• Lock & key?

Pyruvate Kinase Lock & Key

Carboxypeptidase

glucose + ATP glucose-6-phosphate + ADP

INDUCED FIT THEORY OF ENZYME ACTION

• Example Hexokinase

Active site

• 3 dimensional shape on the surface of the enzyme

• Contains specific amino acids to bind the substrate

Induced Fit

• Substrate binding itself changes the shape of the protein

• INDUCED FIT

Induced fit

• The change in the shape of the hexokinase has 2 functions– Changes shape of ATP binding site so it

can bind ATP• This prevents hexokinase acting

independently as an ATPase

– Brings the two substrate binding sites closer, facilitating transfer of phosphate between the two molecules.

CONTROL OF ENZYME ACTION

• Important not to waste valuable cell resources– Prokaryotic cells

• enzyme synthesis is major control mechanism

• e.g. Jacob Monod Hypothesis of enzyme regulation (lac operon)

• Eukaryotic cells more complex

CONTROL OF ENZYME ACTION in EUKARYOTICE CELLS

• Regulate transcription rate of enzyme gene – same as Jacob Monod

• pH e.g. lysosomes pH 5• Modify shape of protein itself

– Inhibitors– Allosteric mechanisms– Covalent modifications– End-product inhibition

Max reached because the active sites are full all the time

COMPETITIVE INHIBITORS

– Inhibitor binds (non covalently) to the active site

– Competes with substrate at active site– Rate slows because active site encounters

fewer substrate molecules per second.– Competitive inhibitors have similar structure

to the substrate– Effect can be overcome by adding more

substrate (increases chance of active site encountering substrate - competing out)

Clinical application

• Methanol poisoning– Methanol formaldehyde (toxic)– Enzyme Alcohol dehydrogenase

• Formaldehyde = Blindness & Death (liver failure)

– Enzyme competitively inhibited by ethanol

Non Competitive inhibitors

• Non Competitive Inhibitors (two types)– Reversible bind non-covalently,

reversibly to the enzyme• Alter conformation of enzyme• Not at the active site, not competed out by

substrate• e.g. inhibition of threonine deaminase by

isoleucine, an example of end product inhibition (& allosteric modulation)

Non Competitive Inhibitors

– Irreversible (could be on active site)• Bind covalently, not able to be removed• Alter conformation of enzyme• Permanently inhibit enzyme • e.g. Penicillins inhibit bacterial cell wall synthesis

by non-competitivitvely binding to the enzymes• Aspirin irreversibly binds to an enzyme which

makes inflammatory lipids• Organophosphorous inhibitors of

Acetylchloinesterase

ANTABUSEDISULFIRAM or

Allosteric Regulation

• Allosteric = other (allo) steric (space/site)• Some enzymes have alternative binding

sites to which modulators (positive or negative [non competitive inhibitor] bind)

• They change the protein’s shape.• Allosteric enzymes often have multiple

inhibitor or activator binding sites involved in switching between active and inactive shapes

• allows precise and responsive regulation of enzyme activity

Cooperative substrate binding

• Allosteric or Regulatory enzymes can have multiple subunits (Quaternary Structure) and multiple active sites.

• Allosteric enzymes have active and inactive shapes differing in 3D structure.

Sigmoid Reaction rate curves

• Enzymes with cooperative binding show a characteristic "S"-shaped curve for reaction rate vs.. substrate concentration. Why?

• Substrate binding is "cooperative." • Binding of first substrate at first

active site stimulates active shape, and promotes binding of second substrate.

                                                                  

Covalent Modification

• Phosphorylation– Phosphate added by kinases– Removed by phosphatases

Example Glycogen Phosphorylase

• Enzyme involved in breakdown of glycogen to produce glucose

• Inactive form not phosphorylated• Active form phosphorylated• Phosphorylase kinase adds phosphate

groups (high energy needs)• Phosphorylase phosphatase removes

phosphate groups (low energy needs)• Activity of phosphatase and kinase under

hormonal control

Allosteric modulation glycogen phosphorylase

• Glycogen phosphorylase also has binding sites for glucose, ATP & AMP

• Glucose & ATP – indicate cell has a lot of energy– Both negative allosteric modulators

• Adenosine Monophosphate (AMP – formed by ATP hydrolysis) – indicate cell has little energy– Positive allosteric modulator

Proteolytic Cleavage

• Proteolytic cleavage of a ZYMOGEN – To produce active enzyme e.g.– Trypsinogen trypsin– Prolipase lipase

• Activation is required, otherwise these enzymes would digest the pancreas– Pepsinogen pepsin– This activation occurs through the action of

pepsin itself– Autocatalysis

Protease

END PRODUCT INHIBITION

• Metabolic pathways usually involve a number of steps from precursor to product. e.g.

• Synthesis of isoleucine from threonine•First step catalysed by threonine

deaminase•Allosterically inhibited by end

product – isoleucine•Allows cell to monitor levels of

product and control production rate appropriately

NON COMPETITIVE INHIBITORS

• Bind to area of the protein other than the active site– Alter conformation (shape) of the

protein changing shape of active site/ making oit more difficult for substrate to bind

• Reversible – non-covalently bound, can be diluted out

• Irreversible – covalently bound cannot be diluted out

• Induced fit: Binding of glucose to Hexokinase induces a large conformational change (diagram p. 386). The change in conformation brings the C6 hydroxyl of glucose close to the terminal phosphate of ATP, and excludes water from the active site. This prevents the enzyme from catalyzing ATP hydrolysis, rather than transfer of phosphate to glucose. 

• • It is a common motif for an enzyme active site to be

located at an interface between protein domains that are connected by a flexible hinge region. The structural flexibility allows access to the active site, while permitting precise positioning of active site residues, and in some cases exclusion of water, following a substrate-induced conformational change.

• This is a molecular model of the unbound carboxypeptidase A enzyme. The cpk, or space-filled, representation of atoms is used here to show the approximate volume and shape of the active site. Note the zinc ion (magenta) in the pocket of the active site. Three amino acids located near the active site (Arg 145, Tyr 248, and Glu 270) are labeled.

• This is a cpk representation of carboxypeptidase A with a substrate (turquoise) bound in the active site. The active site is in the induced conformation. The same three amino acids (Arg 145, Tyr 248, and Glu 270) are labeled to demonstrate the shape change.