Lecture 13 Flashcards
Enzymes
o Enhance reactivity to same extent in forward & reverse rxns
o Exert no effect on rxn equilibrium costant
o For man-made catalysts, enhancement factor is <1,000
o For enzymes, enhancement factor > 1021
o Enzymes lower activation energy of a rxn
Enzyme performances affected by mutation
o Rates of substrate binding
o pKa’s of catalytic ionic groups
o metal ion interactions
o rates of product release
o conformation changes
o Isozymes – different proteins catalyzing same reaction (multiple genesdif proteins)
o Isozymes offer special advantages in metabolism
HK and GK catalyze same rxn but HK acts as back up when glucose levels fall
o Some isozymes have a different allosteric activation or inhibition
o Some isozymes are developmentally regulated
Observations on mutation effects
o Most mutations have no effect on catalysis
o Mutations leading to a loss of essential catalytic residues result in complete loss of enzymatic function
o Even conservative changes of essential catalytic residues are often NOT well tolerated
o Effects of many mutations are undiscovered
Alter binding of as-yet unknown regulators
Prevent unwanted protein-protein interactios
Change organelle targeting
Alter protein turnover kinetics
o Change allosteric properties
Shift saturation curve left or right
Weaken or strengthen subunit-subunit interactions
o Alter enzyme polymerization
Usually attended by loss of activity
o Loss of organelle-targeting sequence
Abnormal compartmentation
o Altered posttranslational modification
Abnormal signal transduction
Michaelis Constant and mutation effects
o Km is concentration of substrate where Velocity is half its max (Km= ½Vmax)
o Simple change in Km (even 2-3fold) leads to both minor and major effects
o Any mutation that decreases Km will increase activity
o Any mutation that increases Km will decrease activity
o Any mutation that increases Vmax will increase activity
o Any mutation that decreases Vmax will decrease activity
o Must use michaelis-menton eqn to evaluate effects on both Km and Vm
o V=Vm/(1+Km/[S])
Drug Susceptibility and mutations
o Chronic myeloid leukemia
Clonal hematopoietic stem cell disorder
Chromosome 9:22 translocation
o Results in formation of Bcr-Abl oncokinase
Exhibits higher protein-tyrosine kinase activity
Strongly implicated in the development of CML
o c-Abl protein kinase domain exists in active and inactive conformations
o x-ray crystallography shows imatinib (Gleevec) binds to inactive form, blocking cell motility
o relapse occurs when mutation cause drug resistance
Amino Acid Deletions
o ATP-dependent chloride transporter
o Phenylalanine deletion at position-508
o Found in ~90% of CF patients
o Protein fails to fold and cannot reach membrane
o New agents help CTFR to fold, with partial restoration of chloride transport
