BIOC/DENT/PHCY 230 LECTURE 2

Lactate dehydrogenase pyruvate + NADHlactate + NAD + M and H subunits: 5 isozymes M subunit has a lower affinity for pyruvate i.e. a higher K M M-type predominates in anaerobic tissues

M 4 predominates in muscle and liver This isozyme can tolerate high concentrations of lactate Can either:continue to convert pyruvate to lactate under anaerobic conditions OR recycle lactate to pyruvate

Cori Cycle Lactate produced in skeletal muscle can be recycled in the liver

H 4 predominates in heart (aerobic tissue) H 4 favours lactate to pyruvate Cardiac cells are permeable to lactate This isozyme:has a low K M for pyruvate and lactate AND is allosterically inhibited by high concentrations of pyruvate This prevents intracellular pH dropping to levels which may affect cellular function

Regulation of glycolysis In general: Inhibited by:ATP, pyruvate, fatty acids, ketone bodies, citrate. Co-ordinately regulated with: glycogen metabolism, gluconeogenesis, pentose phosphate pathway, CAC.

Pasteur effect: no O 2 anaerobic yeast O2O2 glucose consumption Increase in G-6-P and F-6-P Decrease in all intermediates from F-1,6-BP

lactate NAD + NADH ATP ADP AMP Oscillations in glycolytic intermediates Start with aerobic yeast culture NADH NAD +

hexokinase phosphofructokinase (PFK) pyruvate kinase Regulatory steps of glycolysis: 3 key control points

Hexokinase: Glucoseglucose-6-phosphate - increased levels of G-6-P signal high levels of ATP and glycolytic intermediates

Pyruvate kinase: Inhibited by:ATP, acetyl-CoA, alanine, glucagon Activated by:fructose-1,6-bisphosphate phosphoenolpyruvatepyruvate + ATP Four subunit enzyme 3 isozymes (L, M, A)

pyruvate kinase +

Phosphofructokinase Key regulatory enzyme of glycolysis Homotetramer: M r = 360,000 reversibly dissociates to dimer (active form) Inhibited by:ATP, citrate pH

ATP inhibition: Allosteric inhibition: increases K low [ATP] PFK in high [ATP] PFK in T-state R T

Citrate inhibition: Citrate enhances allosteric PFK ATP binding High levels of citrate indicate sufficient CAC intermediates  no need to metabolise (waste) glucose

pH inhibition: Intracellular pH drops under anaerobic conditions Caused by a build up of lactate Low pH decreases PFK activity lactate NAD +

PFK activation Activated by:AMP, ADP, F-2,6-BP [AMP] and [ADP] signal low energy state Fructose-2,6-bisphosphate is an alternative product of F-6-P metabolism Low concentrations of F-2,6-BP activate PFK [F-2,6-BP] increases the affinity of PFK for F-6-P - acts through decreasing ATP inhibition of PFK

P PFK-2 PFK-1 [F-2,6-BP] increases as [F-6-P] increases

Why is this important? Glucose Glucose-6-P Fructose-6-P hexokinase PFK - Fructose-1,6-BP Fructose-2,6-BP +

The take home message: Lactate can be recycled as a fuel LDH isoenzymes influence how lactate is processed by different tissues Glycolysis is tightly regulated in accordance with environmental and intracellular conditions There are three key enzymatic reactions involved in the regulation of glycolysis