4 Enzymes

• Metabolites regulate enzymes by: product feedback inhibition, usually of the first unique enzyme in a biosynthetic pathway.

  e.g. regulation by ATP/ADP/AMP of enzymes of central metabolic pathways, these cause feedback loops.
• Regulation via metabolites involves binding of the control molecule to a regulatory site, not the active site. This is allosteric regulation which is non-competitive.
• Threonine is converted to isoleucine by 5 enzymes, the first of which is threonine deaminase. Threonine deaminase is specifically inhibited by isoleucine - the end product which binds at a distinct regulatory site. This is feedback inhibition, with allosteric regulation.
• Some proteins act as 1:1 inhibitors of other proteins.

  e.g. antitrypsin : elastase in the lungs

  e.g. soybean trypsin inhibitor : trypsin
• Some proteins mediate control of enzymes by interpresting other signals

  e.g. calcium ions are versatile cellular regulators

  Calmodulin is a calcium-binding protein

  Calcium binding by calmodulin changes its conformation activating it

  Activated calmodulin binds to many enzymes and other proteins, modifying their enzymes
• Many enzymes are regulated by phosphorylation, this is a reversable covalent modification. This regulation involves the attachment of a phosphate group to -OH groups.
• Phosphate can be removed by hydrolysis, catalysed by a phosphatase.
• The addition of a phosphate is done by a kinase (EC 2).
• The Kinase cascade: amplifying a signal. This has two start points:

  1. A ligand binds to a receptor protein, which activates a G protein, which activates adenylate cyclase, which activates cAMP, which activates Ras.

  2. A growth factor binds to the PDGFR family kinase, activates a kinase domain on the cytoplasmic side of the membrane which auto phosphorylates itself, activating Ras.

  Ras activates Raf which are ser/thr kinases which phosphorylates MAPKKs which phosphorylate MAPKs. Transcription factors are then phosphorylated to regulate the expression of specific genes.
• Ras is mutated in about 20-25% of human cancers
• BRAF is found mutated in 80% of malignant melanomas - most of the mutations are in the kinase domain and lead to elevated or constitutive activity (active all the time)
• Ca2+ and calmodulin activate:

  1. Ca2+ calmodulin protein kinase
  2. Phosphorylase kinase or glycogen synthase kinase 2
• Kinases and phosphatases are often regulated in turn by other enzymes or products. These are control networks.
• Acetyl CoA, Acetylation of Histones for DNA packing; transcription is a covalent modification
• Myristoyl CoA, Myristoylation of Src for signal transduction
• Farnesyl pyrophosphate, farnesylation of Ras for signal transduction
• Myristoylation and farnesylation are the additions of a carbon chain, a hydrophobic extension anchoring it to the membrane/a sight where it will be active.
• Ubiquitin, ubiquitination of cyclin for cell cycle control, plant hormone signalling, development and gene expression
• Some enzymes are made in an inactive form, cleavage of specific peptide bonds allows activation, this is controlled proteolysis. Complex cascades of proteolytic activation have evolved. Mammalian digestive enzymes are an example, they start as zymogens.
• How chymotrypsin is made:

  Chymotrypsinogen is 245 aa long and synthesised in the pancreas, trypsin makes a covalent disulphide bond at aa 15 and 16. Then chymotrypsin removes a couple small peptides and creates an additional disulphide bond. Creating the 3 chained molecule chymotrypsin.
• Enzymes are not rigid and con covert between alternative conformations, exploited for induced fit and regulation.
• The induced fit model provides enhanced specify since inauthentic mimics do not trigger the conformational change.
• Aspartate transcarbamoylase (ATCase) is the classic example of allosteric regulation
• ATCase catalyses conversion of aspartic acid and carbamoyl-phosphate to carbamoyl-aspartate.

  The first unique step in synthesis of pyrimidines with cytidine triphosphate (CTP) as the product.

  ATCase is allosterically inhibited by the end product CTP
• allosteric regulation does not follow Michaelis-Menten kinetics
• allosteric regulation shows a sigmoidal curve of substrate binding which gives fine control of activity/cooperativity.
• ATCase is a trimer of dimers
• ATP shifts the ATCase curve to the left and reduces co-operativity.

  CTP shifts the curve to the right. Reducing production of ATCase.
• PALA is a competitive inhibitor of ATCase, it has a stable carboxyl group and will not break down into products
• PALA binds at the active site and structurally mimics the transition state, competitive inhibitor of ATCase
• As PALA is stable the protein structure is captured in flagrante. It shows there is a conversion to an alternative subunit arrangement - allosteric regulation
• PALA traps the protein mid-way in its reaction - induces conversion to an alternative subunit arrangement. The protein have a T state (tense) and a R state (relaxed). R state is favoured by substrate binding.
• CTP binding promotes the less active T state conformation, hence CTP inhibits ATCase this is end-product pathway feedback inhibition
• ATCase has two distinct quaternary forms (diff arrangement of subunits)
• The enzyme binds in equilibrium between T and R forms
• Binding of S or an allosteric activator (ATP) shifts the equilibrium towards the R state, binding of an inhibitor (CTP) shifts it towards the T state.
• pro-enzyme = zymogens
• Allosteric regulation exploits proteins as switchable devices
• Cooperativity between multiple identical active sites allows switching from inactive to active conformation and fine regulation of activity over narrow range of substrate conc.
• All effects operate through a conformational change that alters catalytic ability of active site