Recap Test 1 Flashcards
Glycolysis
In the preparatory phase of glycolysis, the hexose molecule ___________________ is phosphorylated at carbons ___ and ___ and then cleaved to form two molecules of __________________________.
Glucose
C1
C6
Glyceraldehyde-3-phosphate
Glycolysis
The conversion of glucose-6-phosphate to fructose-6-phosphate plays a critical role in the overall chemistry of the glycolytic pathway. Why?
Because the rearrangement of the carbonyl and hydroxyl groups at C-1 and C-2 is a necessary prelude to the next two steps: (1) the formation of fructose-1,6-bisphosphate requires the carbonyl group at C-1 be converted to an hydroxyl group and (2) the following step requires a carbonyl group at C-2 for the cleavage of the bond between C-3 and C-4
Glycolysis
The phosphohexose isomerase reaction uses general acid base catalysis to convert glucose-6-phosphate to fructose-6-phosphate via an _________________ intermediate.
Enediol
Glycolysis
Aldolase, which cleaves fructose-1,6-bisphosphate, requires two critical amino acid residues for functioning. What are they?
Lysine
Aspartate
Glycolysis
What is an overview of the aldolase mechanism?
- The carbonyl reacts with active site lysine residue to form an amine, which is then converted to an imine or Schiff base
- Condensation occurs
- Bond cleavage releases G3P
- The resulting enamine covalently linked to the enzyme is isomerized to a protonated Schiff base
- Hydrolysis of the Schiff base generates DHAP
Glycolysis
What is the mechanism of aldolase?
- Lys attacks carbonyl and protonates O
- Hydrogen atom of Lys “kicks back,” forms double bond between N and C, and releases H2O
- Asp deprotonates O on C4, breaking C3 and C4 bond and yielding G3P and an ENAMINE intermediate
- Lone pair of Lys “kicks back” and protonates alkene (C2=C3) via Asp
- Water deprotonates and attacks the carbon of the enamine
- Hydroxyl group “kicks back,” forming a carbonyl group that expels Lys and forms DHAP
Glycolysis
What is the name of this strucutre?
Dihydroxyacetone phosphate
Glycolysis
What is the name of this structure?
Glyceraldehyde-3-phosphate
Glycolysis
What is the mechanism for GAP dehydrogenase?
Oxidation-reduction
Glycolysis
What cofactor does GAP dehydrogenase require?
NAD+
Glycolysis
What amino acid residue acts as the nucleophile in GAP dehydrogenase catalysis?
Cysteine
Glycolysis
What is the mechanism for glyceraldehyde-3-phosphate (GAP) dehydrogenase?
- Cys acts as a nucleophile and attacks carbonyl C (C1)
- Hydride released and forms NADH
- Inorganic phosphate attacks the carbonyl C (C1) and expels enzyme
Glycolysis
Why is GAP dehydrogenase important in glycolysis?
Because it catalyzes the formation of NADH
Glycolysis
GAP dehydrogenase requires the cofactor NAD+ and what other molecule to form 1,3-bisphosphoglycerate from glyceraldehyde-3-phosphate?
Inorganic phosphate
Glycolysis
Phosphoglycerate mutase requires a phosphorylated amino acid residue in its active site to function. What amino acid residue is it?
Histidine
Glycolysis
What is of phosphoglycerate mutase?
- Deprotonated His abstracts proton from hydroxyl on C2
- O acts as nucleophile and attacks electrophilic phosphorus on phosphohistidine residue
- Dephosphorylated His attacks phosphate group on C3, restoring phosphorylated status, and O picks up proton from other protonated His residue
Glycolysis
What intermediate produced via phosphoglycerate mutase is also important in regulating hemoglobin?
2,3-bisphosphoglycerate
Glycolysis
What is the mechanism of action for enolase?
Dehydration and general-acid catalysis
Glycolysis
Enolase requires a metal ion. What metal ion does it require?
Mg2+
Glycolysis
What is the role of Mg2+ in the enolase mechanism?
It makes the proton even more acidic
Glycolysis
What is the point of forming phosphoenolpyruvate?
It is a high energy compound that can be used to make ATP - 2-phosphoglycerate cannot
Glycolysis
What is the mechanism for enolase?
- Lys abstracts proton from C2, forming unstable carbanion
- Lone pair delocalizes to O atom of carbonyl, which is stabilized by Mg2+
- O “kicks back” and expels water from C3 by deprotonating Glu
- Phosphoenolpyruvate is formed
Glycolysis
What two amino acid residues are required for enolase activity?
Lysine
Glutamate
What is the limiting reagent in glycolysis?
NAD+
Fermentation
In alcoholic fermentation, pyruvate must first be decarboxylated into acetaldehyde and carbon dioxide by what enzyme?
Pyruvate decarboxylase
Fermentation
Alcoholic fermentation requires two steps. What are they?
- Decarboxylation of pyruvate to acetaldehyde and carbon dioxide
- Reduction of acetaldehyde to ethanol
Fermentation
What coenzyme is required for the functioning of pyruvate decarboxylase?
Thiamine pyrophosphate or TPP
Fermentation
What part of the coenzyme thiamine pyrophosphate or TPP is the “business end” or the chemically reactive part regarding the decarboxylation of pyruvate?
The thiazolium ring
Fermentation
What is the mechanism for pyruvate decarboxylase?
- Thiazolium ring is deprotonated
- Carbanion functions as nucleophile and attacks the carbonyl C of pyruvate, reducing carbonyl to hydroxyl
- O “kicks back,” releases carbon dioxide, and forms C=C, causing electrons to move on to N of thiazolium ring
- Lone pair “kick back” and double bond between thiazolium ring and C is protonated, leaving behind a hydroxyl group
- Hydroxyl group loses its H, “kicks back” and reforms the carbonyl, and expels enzyme, leaving behind acetylaldehyde
Fermentation
Does the non-enzymatic decarboxylation of an alpha-keto acid work? Why or why not?
No, it does not work; non-enzymatic decarboxylation will only occur with a beta-keto acid as the products of decarboxylation of an alpha-keto acid are unstable
Fermentation
What enzyme reduces acetaldehyde to ethanol in alcoholic fermentation?
Alcohol dehydrogenase
Fermentation
What metal ion is required for alcoholic fermentation?
Zinc is required in the reduction of acetaldehyde to ethanol by alcohol dehydrogenase
What is the first bypass reaction in gluconeogenesis?
Pyruvate is transported into the mitochondrial matrix via pyruvate transporter and acted upon by pyruvate carboxylase with the help of biotin, its covalently attached coenzyme
Pyruvate is first transported from the cytosol into mitochondria where pyruvate carboxylase, a mitochondrial enzyme that requires the coenzyme ____________, converts pyruvate to oxaloacetate. The reaction involves the coenzyme as a carrier of activated ______________.
Biotin
Bicarbonate
What are the two steps to the pyruvate carboxylase mechanism?
- Bicarbonate activation & carboxybiotinyl-enzyme formation: bicarbonate is activated via phosphorylation (at expense of ATP); biotin attacks C carbonyl, which releases inorganic phosphate and leaves carbon dioxide
- Oxaloacetate formation: carbon dixode is carried to next catalytic site, where it is released and reacts with pyruvate, forming oxaloacetate and regenerating the biotinyl-enzyme.
Is the conversion of pyruvate to oxaloacetate via pyruvate carboxylase energy-requiring or energy-producing?
Energy-requiring; it requires ATP
What reactant is required for pyruvate to be converted to oxaloacetate via pyruvate carboxylase?
Carbonate
How is biotin covalently linked to pyruvate carboxylase?
Via an amide linkage at a lysine residue
What is the mechanism for pyruvate carboxylase?
- O on bicarbonate attacks gamma P of ATP, releasing ADP and a proton (“activated molecule of carbon dioxide”)
- N of biotin attacks C carbonyl, ultimately “kicking out” phosphate, which is a good leaving group
- H from pyruvate’s methyl group deprotonates, forming carbanion that attacks the carbonyl C of carboxy-biotin, ultimately kicking out biotin and reforming the enzyme by protonating N on its way out
How many active sites does biotin have?
Two - in one active site, biotin picks up carbon dioxide; in the other active site, biotin transfers carbon dioxide
The product of catalysis by pyruvate carboxylase is oxaloactetate, which must be moved back into the cytosol to continue gluconeogensis. How does the cell accomplish this?
Through the malate-oxaloacetate shuttle system
Identify this compound.
Oxaloacetate
How does the malate-oxaloacetate shuttle system function?
- Oxaloacetate is converted to malate via malate dehydrogenase
- Malate is transported out of the mitochondria via the malate transporter
- Once in the cytosol, malate is converted back into oxaloacetate by malate dehydrogenase
Once oxaloacetate moves back into the cytosol it is acted up by which enzyme?
PEP carboxykinase
Oxaloacetate is converted to _________________________ via PEP carboxykinase.
Phosphoenolpyruvate
What is the mechanism of PEP carboxykinase?
It is a decarboxylation followe by a phosphoryl transfer (from GTP)
What cofactor is required for PEP carboxykinase to function?
GTP
What chemical changes does pyruvate carboxylase catalyze? In other words, what happens to pyruvate?
A carbon dioxide molecule (COO-) is added to pyruvate, resulting in a four carbon molecule with an additional terminal COO- group
Does PEP carboxykinase facilitate a carboxylation or decarboxylation?
Decarboxylation
What is the first step in the PEP carboxykinase mechanism?
Removal of CO2
What is the second step in the PEP carboxykinase mechanism?
Phosphoryl transfer from GTP
PEP carboxykinase catalyzes the conversion of oxaloacetate to what intermediate of glycolyis?
Phosphoenolpyruvate
Describe the mechanism of PEP carboxykinase?
- O anion on C4 kicks back and expels CO2
- Carbanion forms on C3, which then kicks back, forming an alkene with C2, and the carbonyl oxygen becomes phosphorylated
Note that the carbon dioxide added to pyruvate in the _____________ ___________________ step is the same molecule that is lost in the _______ _______________ reaction. This carboxylation-decarboxylation sequence represents a way of “activating” pyruvate, in that the decarboxylation of oxaloacetate facilitates PEP formation.
Pyruvate carboxylase
PEP carboxykinase
