Biochem Exam 4 (enzymes)

Hexokinase

• Step 1 of glycolysis

• Converts glucose to G6P

• Allosteric inhibition by its product (G6P)

• Operates near Vmax

  • always active when glucose is present

Glucokinase (GK)

• the glucose sensor

• high Km → only active at high glucose concentrations

• regulated by glucokinase regulatory protein

Phosphoglucose Isomerase

• Step 2 of glycolysis

• converts aldose to ketose

• positions C1 for phosphorylation in step 3

• prepares molecule for symmetric cleavage in step 4

Phosphofructokinase-1 (PFK-1)

• Step 3 of glycolysis

• converts Fructose-6-phosphate → fructose-1,6-bisohosphate (F-1,6-BP)

• rate-limiting step of glycolysis (irreversible)

• Major site of allosteric regulation

  • activators

    • AMP

    • ADP

    • F-2,6-BP

    • Pi

  • Inhibitors

    • ATP

    • Citrate

    • Low pH

• Master regulator → F-2,6-BP

Aldose reaction

• Step 4 of glycolysis

• converts F-1,6-BP into DHAP and G-3-P

• does this by schiff base intermediate with lys (animals) or ZN2+ as cofactor (bacterial/fungi)

Triose Phosphate Isomerase

• Step 5 of glycolysis

• Converts DHAP into G3P

• Kinetically perfect enzyme

  • reaction rate is only limited by diffusion

  • Kcat/Km reaches theoretical maximum

Glyceraldehyde-3-bisphosphate dehydrogenase (GAPDH)

• Step 6 in glycolysis

• converts G3P into 1,3-bisphosphoglycerate + NADH + H+

• Oxidation of aldehyde to carboxylic acid

• Phosphorylation of inorganic phosphate bond

• Involves covalent catalysis and a nicotinamide coenzyme

Phosphoglycerate kinase

• Step 7 of glycolysis

• Converts 1,3-bisphosphoglycerate + ADP to 3-phosphoglycerate + ATP

• Substrate-level phosphorylation

Phosphoglycerate Mutase

• Step 8 of glycolysis

• Converts 3-phosphoglycerate to 2-phosphoglycerate

• Mutase = catalyze migration of a functional group within a substrate class

• Phosphoryl group transfer from C3 to C2 through a phospho-histidine intermediate

Enolase

• Step 9 of glycolysis

• converts 2-phosphoglycerate to phosphoenolpyruvate (PEP) and water

• Dehydration reaction

Pyruvate Kinase

• Step 10 of glycolysis

• Converts PEP to pyruvate + ATP

• Second substrate-level phosphorylation

• Regulation

  • activated by F-1,6-BP and AMP

  • Inhibited by ATP and Acetyl-coA

• Mechanism is two steps

  • Phosphoryl transfer (PEP → ATP)

  • Enol-Pyruvate formed (Unstable)

• Different regulators depending on the tissue type

Pyruvate Dehydrogenase

• Step 0 of TCA

• Location: Mitochondrial matrix

• converts Pyruvate + CoA + NAD+ into Acetyl-coA + CO2 + NADH

• Made up of three enzymes

  • Pyruvate dehydrogenase (TPP cofactor)

  • Dihydrolipoyl transacetylase (Lipoate)

  • Dihydrolipoyl dehydrogenase (FAD, NAD)

• 5 coenzymes required

  • TPP - thiamine pyrophosphate

  • Lipo - Lipoate

  • FAD - Flavin adenine dinucleotide

  • CoA - coenzyme A

  • NAD - nicothiamide adenine dinucleotide

• Regulation

  • Allosteric regulation

    • Inhibitors: Acetyl-CoA; NADH; ATP

    • Activators: CoA; NAD+; ADP

  • Covalent modification

    • PDH Kinase → phosphorylates E1 → Inactive

    • PDH phosphotase → Dephosphorylates E1 → Active

  • Hormonal control

    • Insulin → activates phosphotase → PDC on

    • Glucagon → activates kinase → PDC off

Citrate synthase

• Step 1 of TCA

• Converts acetyl-coA + oxaloacetate + H2O to Citrate + CoA-SH

• Condensation reaction

• Regulation

  • Inhibited: ATP; NADH; Succinyl-CoA; Citrate

  • Activated: ADP

Aconitase

• Step 2 of TCA

• Converts Citrate to isocitrate via cis-aconitate

• Dehydration then rehydration

• Moves -OH group from tertiary to secondary carbon = prepares molecule for oxidative decarboxylation

Isocitrate dehydrogenase

• Step 3 of TCA

• converts isocitrate to alpha-ketoglurate

• First oxidative decarboxylation

• First NADH of the cycle

• First CO2 released

• Oxidation of a secondary alcohol to a ketone

• Regulation

  • inhibited by: ATP, NADH

  • Activated by: ADP, Ca2+