BIOC/DENT/PHCY 230

LECTURE 2

Lactate dehydrogenase

pyruvate + NADH lactate + NAD+

M and H subunits: 5 isozymes

M subunit has a lower affinity for pyruvate

i.e. a higher KM

M-type predominates in anaerobic tissues

M4 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

H4 predominates in heart (aerobic tissue)

H4 favours lactate to pyruvate

Cardiac cells are permeable to lactate

This isozyme: has a low KM 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 O2

anaerobic yeast

O2

glucose consumption

Increase in G-6-P and F-6-P

Decrease in all intermediates from F-1,6-BP

lactateNAD+

NADH

ATP

ADPAMP

Oscillations in glycolytic intermediates

Start with aerobic yeast culture

NADHNAD+

hexokinase

phosphofructokinase

(PFK)

pyruvate kinase

Regulatory steps of glycolysis:

3 key control points

Hexokinase:

Glucose glucose-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

phosphoenolpyruvate pyruvate + ATP

Four subunit enzyme

3 isozymes (L, M, A)

pyruvate kinase

+

Phosphofructokinase

Key regulatory enzyme of glycolysis

Homotetramer: Mr = 360,000

reversibly dissociates to dimer (active form)

Inhibited by: ATP, citrate

pH

ATP inhibition:Allosteric inhibition: increases KM

@ low [ATP] PFK in R-state

@ 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

lactateNAD+

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-BPFructose-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