Previous lecture:
•Chemical catalysis-stabilization of transition state
•Acid-base catalysis, Electrophilic catalysis, Covalent catalysis, Metal ion catalysis
•Importance of pKa values
Today: •Carbonic anhydrase •PLP – mediated catalysis Metal ions as Covalent Catalysts- Carbonic anhydrase
- + CO2 + H2O HCO3 +H Metal ion covalent catalysis- Carbonic anhydrase Metal ion covalent catalysis- Carbonic anhydrase
- + CO2 + H2O HCO3 +H
Coenzyme Catalysis
Coenzyme (Cofactor) small organic molecule or (metal ion) essential to the catalytic action of an enzyme Common cofactors: FMN flavin mononucleotide FAD flavin adenine dinucleotide NAD+/NADH nicotinamide adenine dinucleotide NADP+ / NADPH nicotinamide adenine dinucleotide phosphate PLP pyridoxal 5’-phosphate PMP pyridoxamine 5’-phosphate THF tetrahydrofolate CoASH coenzyme A GSH glutathione ATP adenosine triphosphate Pyridoxal Phosphate (PLP) Enzymology
•PLP is the modified form of Vitamin B6 (pyridoxal) •PLP is a coenzyme heavily involved in amino acid transformation reactions •Central to its mechanism is the formation of a Schiff base with the enzyme (through a covalent bond with Lys) •Schiff base is formed through the condensation of an amine with a carbonyl group. •Serves to stabilize carbanion intermediates of amino acids Pyridoxal Phosphate (PLP) Enzymology
PLP most commonly involved in decarboxylation or transamination reactions at the α-Carbon of amino acids
HO O H O OH OH HO O P O
N Me O N Me
vitamin B6 PLP Pyridoxal Phosphate (PLP) Enzymology Pyridoxal Phosphate (PLP) Enzymology
•PLP-requiring enzymes act via the formation of a planar Schiff base between the amino acid to be modified (substrate) and the coenzyme Pyridoxal Phosphate (PLP) Enzymology •PLP-requiring enzymes act via the formation of a planar Schiff base between the amino acid to be modified (substrate) and the coenzyme •That leads to the formation of a resonance-stabilized carbanion with a quinoid structure •The key to catalysis is the electrophilic nitrogen of the pyridine ring - draws electrons away from the substrate and stabilizes the carbanion intermediate (electron sink) Aspartate Aminotransferase active site with PLP
Dr. Jack Kirsch-Berkeley
Next Class
Proximity effect Steady state kinetics