Metal ion catalysis pyruvate kinase (padhmasri)

This topic is about metal ion catalysis with the example of pyruvate kinase enzyme which is involved in glycolysis

1. Metal ion catalysis pruvate kinase Presentation by, K.PADHMASRI MSC.BIOCHEMISTRY

2.  Metal ion catalysis -Two classes of metal ion dependent enzymes 1-Metalloenzymes  Enzymes that contain tightly bound metal ions are termed –  Metalloenzymes Metal ion tightly bound to the enzyme(covalent bond) ( Fe 2+ , Fe 3+ , Cu 2+ , Zn 2+ , Mn 2+ )   This metal ion is an essential portion in the enzyme , if removed , there will be no reaction .

3. 2-Metal activated enzymes Enzymes that require metal ions as loosely bound cofactors    are termed as metal-activated enzymes loosely bond metal ions (alkali or alkaline metal including Na + , K + , Mg +2 , Ca 2+ .  if the metal ion is removed from the reaction , the enzyme is still acting but in low level .  Metal ions enhance catalysis in three major ways : 1- Binding to and orienting substrates for reaction as Mg 2+ binding to ATP . 2-Mediating redox reaction through changes in oxidation state such as reduction of O2 to H2O through electron transfer  3-Electrostatic stabilization or shielding of negative charges as Mg2+ binding to ATP .

4. Pyruvate kinase Pyruvate kinase is an enzyme that catalyzes the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP in glycolysis and plays a role in regulating cell metabolism

5. Mechanism of pyruvate kinase Pyruvate kinase catalyzes the direct transfer of phosphate from phosphoenolpyruvate (PEP) to ADP to produce ATP and pyruvate .

6. stages of pyruvate kinase cataysis Step 1: The phosphoryl transfer from phosphoeno l pyruvate to ADP afford the magnesium stabilised enolate of pyruvate ( enol pyruvate ). step 2 : The ketonization of enolpyruvate to pyruvate by addition of proton

7. Pyruvate kinase catalyzes the direct transfer of phosphate from phosphoenolpyruvate (PEP) to ADP to produce ATP andpyruvate . This reaction is favorable due to the high energy of hydrolysis of PEP . During catalysis, the active site is occupied by both substrates (PEP and ADP, which is complexed with Mg 2+ ), one monovalent cation and one additional enzyme-bound divalent cation Pyruvate involves a cyclic metal bridge complex, ACTIVATION OF ALKALINE EARTH METAL CATIONS (Mg2 +) Pyruvate kinase is tetrameric enzyme ,which require Mg2+, all of which bind to region of active site. Mg2+ reduces the electrostatic repulsion between phosphodonor (PEP) and the nucleophile (beta phosphorus group of ADP)

8. Activation of alkali metal cation K+ K+ binding to large negative group of an inactive form of enzyme and involved in acquisition of active conformational changes of enzyme to a more active form. K+ coordinate the phosphoenol pyruvate carboxyl group and in the presence of K+ , the affinity of PK-Mg2+ to PEP and to ADP-Mg2+ Which facilitate the progress of reaction via E-Mg2+-PEP COMPLEX also known as metal bridge complex

9. STEP:1

10. Enzyme bound cation probably binds to water forming a co- ordinated hydroxyl group which can attack the phosphoenol pyruvate

11. MECHANISM OF PYRUVATE KINASE

12. step 2 : The ketonization of enolpyruvate to pyruvate by addition of proton Transfer of the phosphate from PEP to ADP leaves the energetically less-stable enol form of pyruvate bound in the active site Tautomerization of enolpyruvate to the more stable keto form of pyruvate contributes to the favorable energetics of phosphate transfer from PEP to ADP. Tautomerization occurs when enolpyruvate accepts a proton , from a water molecule that is held in position by conserved active site residues (T328 and S362 in humans catalysis the products leave the active site, and neither substrate binding nor release of products is thought to be ordered .

13. Enol pyruvate ( enol ) TAUTOMERISATION/ Pyruvate ( ketone )