Glycolysis - Regulation Lecturer: Rick Kahn RRC G-217 Phone: Objectives: To begin to think about enzymes as regulated catalysts To understand the different ways enzymes can be regulated To learn the key, regulated steps in glycolysis, the mediators of regulation, and how it is connected to other pathways So today we will talk first about general features of metabolic/enzyme regulation and then the specifics as they relate to glycolysis.

Glycolysis is just one of many pathways all going on in a cell YOU ARE HERE

10 enzymatic steps get you from glucose to pyruvate Phosphorylation of glucose and fructose 6-phosphate require input of 2 ATPs Cleavage of fructose 1,6 bisphosphate (hexose) yields two trioses, only one of which is further metabolized in glycolysis but the two are interconvertible When bookkeeping, double everything after aldolase Two steps yield ATP (x2=4), one step reduces NAD + to NADH The Glycolytic Pathway

Hypothetical Metabolic Pathway In enzyme-limited steps the substrate accumulates In substrate-limited reactions the substrate is quickly metabolized so does not accumulate Regulation can only occur at sites that are enzyme-limited

Enzymes are REGULATED catalysts Regulation can occur as a result of: limiting substrate availability - OFTEN limiting enzyme concentration – Genetic disease? limiting turnover rate – different isoforms? covalent modification of the enzyme (e.g. phosphorylation)—often related to endocrine control through interaction(s) with modulators that bind to the substrate binding site (competitive inhibition) to the product binding site (product inhibition) or another, termed allosteric, site, that can be stimulatory or inhibitory – Key concept

Catalytic sites on enzymes “flicker” between having a substrate and a product binding sites The Enzyme The Substrate The Product

“Simple” catalysis Substrate Product Enzyme in substrate binding conformation Enzyme in product binding conformation Chemistry Happens Here

Competitive inhibition Substrate Inhibitor Note: Inhibitor can be an alternate substrate or simply bind to the catalytic site

Competitive inhibition SubstrateInhibitor Note: Inhibitor can be an alternate substrate or simply bind to the catalytic site

Product Inhibition: Excess product “ties up” the enzyme in one state and thus slows catalysis

Allosterism Alteration of the activity of a protein (as an enzyme) by combination with another substance at a point other than the chemically active site. ---Webster’s 9 th Collegiate Dictionary The influence to an enzyme activity brought about by the change in conformation of the protein in response to the binding of a substance or other effector at a site other than the active site. ---Stedman’s Medical Dictionary 26 th edition

Allosteric inhibition: Binding of a ligand outside the catalytic site—can activate OR inhibit Substrate Allosteric Activator Allosteric Inhibitor “Simple” Enzyme Enzyme with Allosteric activating site Enzyme with Allosteric activation and inhibitory sites Enzyme with Allosteric inhibitory site

Allosteric activation K D for substrate 10 μM K D for substrate 0.10 μM

Allosteric Inhibition In the absence of the allosteric inhibitor, activity is high In the presence of allosteric inhibitor, activity is low; substrate can accumulate

Highly regulated (important) enzymes can have both allosteric activator and inhibitor sites High Activity Low Activity Moderate Activity

A highly specialized (rare) case: activator and inhibitor sites overlap (PFK1) Activator e.g., ADP Gamma phosphate of ATP PFK1 Inhibitor: ATP High Activity Low Activity

Which steps in a pathway are the ones you would want to regulate? Entry to the pathway (the committed step)--PFK1 Exit from a pathway--Pyruvate Kinase Entry/exit points within one pathway that go to or come from another pathway--Hexokinase (kind of)

Glucose transporters simply facilitate diffusion (0 energy req’d) Transport would be readily reversible BUT 1)Glucose constantly being metabolized 2)Rapid phosphorylation prevents leakage out

Hexokinase and Glucokinase each catalyze the same reaction but… Hexokinase is subject to product inhibition but glucokinase is not When glycolysis (PFK1) is inhibited, the pathway gets constipated and backs up, resulting in shut down of hexokinase Brain Muscle Liver

The primary site of regulation of the glycolytic pathway is PFK1 Commitment to Glycolysis

Fructose 6-phosphate Fructose 1,6-bisphosphate Phosphofructokinase 1 (PFK1) Glucose 6-phosphate Glycolysis Pyruvate TCA cycle Citrate ATP Work ADP + _ _ PFK 1 can sense the “energy charge” of the cell because both ATP (inhibitory) and ADP (stimulatory) are allosteric modulators Regulators of PFK1 Adenine nucleotides (ATP, ADP) Citrate F2,6 bisphosphate pH

Phosphofructokinase 2 (PFK2) generates fructose 2,6- bisphosphate, an allosteric activator of PFK1 Fructose 6-phosphate Fructose 1,6-bisphosphate PFK1 Glucose 6-phosphate Fructose 2,6-bisphosphate PFK2 + Glycolysis Fructose 2,6-bisphosphate is a dead end and can only be cleaved by phosphatase back to fructose 6-phosphate Why, why, why? Why is there PFK2 and fructose 2,6 bisphosphate? Answer: PFK2 is the supercharger of glycolysis. Fructose 2,6-bisphosphatase

PFK2 allows for endocrine (insulin and glucagon) regulation of glycolysis

Last step (Pyruvate Kinase) is also regulated by glucagon and F1,6BP When [Glucose] BLOOD is low, glucagon is up, PKA is active, liver PK gets phosphorylated, and is turned off— this is part of the switch to gluconeogenesis Fructose 1,6 bisphosphate is an allosteric activator of PK… and… ATP is an allosteric inhibitor

Summary of Endocrine Regulation (of carbohydrate/glucose metabolism) Insulin and Glucagon are the main endocrine factors (each is a protein hormone) that modulate blood glucose levels and they act antagonistically The Pancreas secretes Insulin (  cells) and Glucagon (  cells) Insulin: In liver it  production of glucose by  gluconeogenesis and glycogenolysis In muscle and liver it  glycogenesis In muscle and fat cells it  uptake of glucose Glucagon: In liver it  glycogenolysis and gluconeogenesis

Summary of Glycolysis Regulation Three enzymes involved are: PFK1, PK, and hexokinase (and PFK2) The changes all make sense. –When there is plenty of ATP in the cell, slow it down to allow storage of glycogen in the liver; thus hexokinase off, PFK off (ATP and citrate allosteric inhibition, little ADP to activate). –When blood glucose is high, insulin is up, PFK1 is on, PFK2 is on (further activating PFK1), PK is on –When blood glucose is low, most cells go into “energy sparing” mode to conserve for brain/muscle; glucagon is up, PFK1 is off, PFK2 is off, PK is off

Regulation of input and output from glycolytic pathway integrates it with other metabolic pathways REGULATED Note: metabolism of fats feeds into TCA cycle downstream of pyruvate – this is why it is important to have citrate as an allosteric inhibitor of glycolysis