Biochemistry- Enzymes/Reactions Flashcards
Glycosidase
Breaks glycosidic bonds
AKA Glycoside hydrolase
Iduronate sulfutase deficiency
Impaired degradation of GAGs (Dermatan sulfate and heparan sulfate affected)
- Results in Hunter Syndrome (MPS II)
α- L iduronidase deficiency
Impaired degradation of GAGs (Dermatan sulfate and heparan sulfate affected)
- Results in Hurler Syndrome (MPS I)
Sanfilippo syndrome mechanism
- Missing enzyme for one of four steps to remove N-Sulfated/N-acetylated glucosamine residues from HEPARAN SULFATE
Depending on type, missing: A- Heparan sulfamidase B- N-acetyl glucosaminidase C- Glucosamine- N- Accetyl transferase def. D- N-acetylglucosamino-6-sulfutase
ß- Glucuronidase definciency
Impaired degradation of GAGs (Dermatan sulfate and heparan sulfate affected)
- Results in Hurler Syndrome (MPS VII)
Lactase deficiency
- Intolerance of ingested milk products (Lactose intolerance)–> diarrhea, bloating, flatulence, increased H2 in breath
Can be congenital or due to intestinal injury
Sucrase-isomaltase deficiency
Ingested Sucrose intolerance
- Impaired split of sucrose, maltose, maltotriose
- -> diarrhea, bloating, increased H2 in breath
Fructose intolerance
Deficiency in GLUT-5
- Can’t transport/absorb fructose (in large/moderate amounts)
- GI distress, gas, H2 gas in breath
NOT the same as inability to metabolize fructose
I-cell disease
Deficiency in ability to phosphorylate mannose 6 (on a glycoprotein)
- Glycoprotein can’t mark target enzymes to go to lysosome for destruction, so there is a bulidup of digestive enzymes in the cell
Reaction catalyzed by hexokinase?
- Cofactors/Requirements (if any)?
D-Glucose –> Glucose-6-phosphate
Irreversible, Step 1 of Glycolysis
- NOT in liver/pancreas
- Requirement: Mg 2+, ATP
Reaction catalyzed by glucokinase?
- Cofactors/Requirements (if any)?
D- Glucose –> Glucose-6-phosphate
Irreversible, Step 1 of glycolysis
- In liver/pancreas ONLY
- Requirement: Mg 2+, ATP
Reaction catalyzed by phosphofructokinase?
- Cofactors/Requirements (if any)?
Fructose-6- Phosphate –> Fructose-1,6-
bisphosphate
Irreversible, Rate limiting, and Committed step of Glycolysis (Step 3)
- Requirement: Mg 2+, ATP
Reaction catalyzed by Glyceraldehyde 3- Phosphate dehydrogenase?
- Cofactors/requirements?
Glyceraldehyde-3-phosphate 1,3-bisphosphoglycerate
Reversible, not regulated (step 6 of glycolysis)
- First NADH generated (x2)
- Requirement: NAD+, Pi
Arsenate (arsenic poisoning) affects this step of glycolysis
Reaction catalyzed by Phosphoglycerate kinase?
- Cofactors/requirements?
1,3-bisphosphoglycerate 3-phosphogylcerate
Reversible, not regulated (step 7 of glycolysis)
** First substrate level phosphorylation, ATP generated (x2)
- Requires ADP & Pi, Mg2+
Reaction catalyzed by pyruvate kinase (PK)?
- Cofactors/requirements?
Phosphoenolpyruvate –> Pyruvate
- Irreversible, regulated (step 10 of glycolysis)
**Substrate level phosphorylation, ATP generated (x2)
-Requires: ADP, Pi, Mg2+, K+
How much ATP and NADH is generated during aerobic glycolysis?
2 ATP, 2 NADH
Reaction catalyzed by lactate dehydrogenase?
- Cofactors/requirements?
Pyruvate –> Lactate (reduction)
- Last step in anerobic glycolysis
Requires: NADH
Lens/cornea, Kidney medulla, RBCs, testes, leukocytes all rely on anerobic glycolysis
Pasteur effect
The slowing of glycolysis in the presence of oxygen (b/c more ATP is produced)
- Glycolysis is faster under aerobic conditions
Reaction catalyzed by Enolase?
- Cofactors/requirements?
2-phosphglycerate PEP
Reversible & not regulated, Step 9 in glycolysis
- Requirement: Mg2+
Water is eliminated
Fluoride inhibits this enzyme, so bacteria in mouth produce less lactic acid, less cavities
Mechanism of Arsenic poisoning?
- Affects glycolysis and TCA
- In glycolysis, Arsenate gets incorporated into glyceraldehyde 3-phosphate –> forming 1-Arseno-3-phosphoglycerate
- Hydrolyses spontaneously/easily to 3-phosphoglycerate b/c unstable
- Bottomline: NO SUBSTRATE LEVEL PHOSPHORYLATION IN STEP 7, NO ATP GAIN, RBCS SUFFER BECAUSE CANT PRODUCE ATP
- In the TCA–>
- Arsenite inhibits enzymes requiring LIPOIC ACID (i.e. PDH, α-ketoglutarate dehydrogenase, branched-chain amino acid α–keto acid dehydrogenase)
- Arsenite forms a stable complex with the thio group of lipoic acid - Affects the brain and cause neurologic disturbance and death
Regulation of glycolysis at step 1?
Inhibition:
- Negative feedback of glucose 6- phosphate (ONLY HEXOKINASE, b/c glucokinase has larger Km and less affinity, and larger V max)
Activation (indirect):
- Insulin stimulates GLUT 4 to come to cell membrane from inside cell, so intake of glucose into adipose (ie) cells increases
- Hexokinase is active at low glucose levels compared to glucokinase
Regulation of glycolysis at step 10?
Allosterically activated:
- Fructose 1,6 bisphosphate (in liver, muscle 1&2, RBC by all 4 isozymes)- FEED FORWARD
Inhibition:
- ATP (allosteric)
- IN LIVER: glucagon decreases PK (pyruvate kinase) activity by phosphorylation
Regulation of glycolysis at step 3?
Activation (allosteric):
- AMP, ADP, Fructose-2,6-bisphosphate
F-2,6-BP is regulated by insulin/glucagon
Inhibition:
- ATP, citrate, high [H+]
Mechanism of Fructose-2,6-bisphosphate?
- A VERY POTENT ACTIVATOR OF PFK-1 (step 3 glycolysis enzyme)
- Also inhibits gluconeogenesis?
-Production is regulated by tandem enzyme: has Phosphofructokinase-2 (PFK-2) domain
AND Fructose-2,6-bisphosphatase (FBP-2) domain.
** In the presence of Insulin–> phosphotase active–> phosphotase dephosphorylates PFK-2–> PFK-2 (w/ATP) catalyzes phosphorylation from F-6-P to generate F-2,6-BP –> more activation of PFK-1 –>more glycolysis
** In the presence of glucagon –> Protein Kinase A is active –> PKA phosphorylates FBP-2–> FBP-2 removes phosphate from Fructose 2,6-BP (inactivates)–> less activation of PFK-1 –> less glycolysis
Pyruvate kinase deficiency
- RBCs can’t complete glycolysis (b/c need lactate to regenerate NAD+)
- 50% less ATP produced
- ATP is not available to regulate ION TRANSPORTERS
- Hemolytic anemia occurs
Reaction catalyzed by Pyruvate dehydrogenase (PDH) complex?
Requirements?
Pyruvate –> Acetyl CoA
Requires:
- Thiamine pyrophospate (Vitamin B1) - E1 aka Pyruvate DEcarboxylase
- Lipoic acid- E2
- CoA- E2
- FAD- E3
- NAD- E3
-CO2 is released (lost when TPP binds to pruvate, E1 domain)
What regulates PDH complex? What influences the regulators?
Inhibition:
* PDH kinase (inhibits E1, less TCA))
- ATP, Acetyl CoA, and NADH activate the inhibitor (enough energy, less TCA)
- Pyruvate INHIBITS the inhibitor (PDH kinase) – not enough energy, more TCA
Activation:
* PDH phosphotase (activates E1)
- Ca2+ released by skeletal muscle cells during contraction activates activator (need more energy, more TCA)
Reaction catalyzed by citrate synthase?
Requirements?
Oxaloacetate –> Citrate (Step 1 of TCA)
Irreversible, regulated
Requires: Acetyl CoA, H2O
Regulation of citrate synthase?
Inhibition: Citrate, NADH, Succinyl CoA
What does fluoroacetate inhibit?
Fluoroacetate is a rat poison/plant toxin
- Inhibits Acitonase (Fe-S) enzyme that catalyzes Citrate to Isocitrate reaction (Step 2 of TCA)
Reaction catalyzed by isocitrate dehydrogenase?
Requirements?
Isocitrate –> alpha-ketoglutarate (step 3 of TCA)
RATE LIMITING, irreversible, regulated
Requires: NAD+
*1ST NADH YIELD, 1ST CO2 RELEASED
Reaction catalyzed by alpha-ketoglutarate dehydrogenase?
Requirements?
alpha-ketoglutarate succinyl CoA (step 4 of TCA)
Requires:
- Vitamin B1 (thiamine)
- Lipoic Acid
- CoA
- FAD
- NAD
*2ND NADH YIELD, 2ND CO2 RELEASED
Regulation of Isocitrate dehydrogenase?
Activation: ADP, Ca2+ (in muscle)
Inhibition: ATP, NADH
Isocitrate –> alpha-ketoglutarate (step 3 of TCA)
Regulation of alpha-ketoglutarate dehydrogenase?
Activation: Ca2+ (muscle)
Inhibition: ATP, NADH, GTP, Succinyl CoA
alpha-ketoglutarate succinyl CoA (step 4 of TCA)
Reaction catalyzed by succinate thiokinase?
Requirements?
Succinyl CoA Succinate (step 5 of TCA, start of OAA regeneration)
Reversible
Requirements: GDP and Pi
**GTP produced
Reaction catalyzed by succinate dehydrogenase?
Requirements?
Succinate Fumarate (step 6 of TCA)
Reversible; Enzyme is in mitochondrial inner membrane (Complex II of ETC)
Requirement: FAD
**1ST AND ONLY FADH2 PRODUCED
Reaction catalyzed by Malate Dehydrogenase?
Requirements?
L-Malate Oxaloacetate (step 8 of TCA)
Reversible; not very favorable but it’s ok b/c step 1 of TCA is very favorable
Requirement: NAD+
**3RD AND FINAL NADH GENERATED, REGENERATION OF OAA SO TCA WILL RESTART
How much ATP via equivalents is produced via TCA?
10-12 ATP (depending on tissue; brain and skeletal m. will produce less b/c they use glycerol phosphate shuffle)
PDH deficiency (E1 deficiency specifically)
Causes congenital lactic acidosis (because pyruvate gets reduced to lactate instead)
- neurologic symptoms b/c brain is sensitive to lactic acidosis
Leigh syndromes (subacute necrotizing encephalomyelopathy)
- Either a PDH deficiency or a PC deficiency (30 possible gene mutations)
Causes lactic acidemia and respiratory failure
Beriberi disease/Wernicke-Korsakoff syndrome (similar mech)
- Thiamine deficiency
cofactor of PDH, Alpha-ketoglutarate dehydrogenase
Reaction catalyzed by Pyruvate carboxylase (PC)?
Requirements?
Regulation?
Pyruvate –> oxaloacetate
Irreversible
- Oxidative step in gluconeogenesis to overcome step 10 in glycolysis
- Also anapleroic reaction for TCA (restore OAA levels)
Requirements: Biotin, ATP (x2), Mg2+, CO2
Takes place in mitochondria
Regulation: allosterically activated by Acetyl CoA
Reaction catalyzed by phosphoenolpyruvate carboxykinase?
Requirements?
Oxaloacetate –> Phopsphoenol pyruvate
Irreversible
- Oxidative step in gluconeogenesis to overcome step 10 in glycolysis
Requirements: GTP (x 2)
- Note: CO2 is released
1/2 mitochondrial, 1/2 cytosolic in humans
Reaction catalyzed by fructose-1,6-bisphasphotase?
Requirements?
Fructose 1,6-bisphosphate –> Fructose 6-phosphate
Irreversible
- Oxidative step in gluconeogenesis to overcome step 3 in glycolysis
Requirements: just H2O
Phosphate released
Regulation of Fructose-1,6-bisphosphatase?
Inhibition: (allosteric) Fructose-2,6-bisphosphate (Insulin/Glucagon), AMP
Reaction catalyzed by glucose 6-phosphatase?
Requirements?
Glucose 6-phosphate –> Glucose
Irreversible
- Oxidative step in gluconeogenesis to overcome step 1 in glycolysis
Requirement: just H2O
Phosphate released
**LIVER AND KIDNEYS ONLY RELEASE FREE GLUCOSE
What are reactions in gluconeogenesis that require ATP?
3-phosphoglycerate 1,3-phosphoglycerate (x 2)
Enzyme: Phosphoglycerate kinase
What are reactions in gluconeogenesis that require NADH?
1,3-phosphoglycerate glyceraldehyde 3-phospate (x2)
Enzyme: glyceraldehyde phosphate dehydrogenase
How much energy/reagents does gluconeogenesis require?
6 ATP, 2 NADH
What are the precursors of gluconeogenesis?
- Lactate (converted to pyruvate by Lactate DeHydrogenase)
- alpha-keto acids and amino acids (from catabolism of glucogenic AAs, form oxaloacetate)
- Glycerol (converted to glycerol-3-phosphate by glycerol kinase –> from hydrolysis of triglycerides)
What states result in gluconeogenesis regulation?
- Fasting
- Prolonged exercise
- High protein diet
- Stress or injury
- Substrate availability (lactate, pyruvate, glucogenic AAs, glycerol)
What is alcohol’s effect on gluconeogenesis?
- Alcohol metabolism reduces NAD+ to NADH (NAD+ is needed for glycerol-P-dehyrogenase and LDH)
- Less NAD+ means less gluconeogenesis and more lactate buildup
- This can result in hypoglycemia and lactic acidosis
Which enzymes in the pentose phosphate pathway particpate in reactions that generate NADPH?
- Glucose 6-P dehydrogenase (G-6-P to 6-phosphogluco-delta-lactone)- 1st oxidative reaction
^(negative feedback from NADPH!!!) - 6-phoshogluconate dehydrogenase (6-phosphogluconate to ribulose 6-phosphate) - 3rd oxidative reaction
Glucose 6-P dehydrogenase deficiency mechanism
- Less NADPH–> less (reduced) glutathione–> more reactive oxygen species -> more damaged hemoglobin –> more hemoglobin aggregation –> Heinz bodies –> weak/fragile RBCs –>hemolysis (hemolytic anemia)
NOTE: TOTAL absence is LETHAL, usually decreased activity
Reaction catalyzed by Glycogen phosphorylase?
Requirements?
Phosphorolysis of glycogen at alpha(1->4) linages using inorganic phosphate donation to oxygen of the glycosidic bond (glycogen breakdown)
One residue at a time
Irreversible, regulated
Requirement: Vitamin B6 (PLP, pyridoxyl phosphate)
Reaction catalyzed by debranching enzyme?
Two functions (glycogen breakdown):
- 4:4 glucan transferase: transfer 3 outer glucoses on branch to non-reducing end of chain
- alpha-1,6- glucosidase removes remaining glucose that is alpha 1,6 linked (releases as FREE GLUCOSE, little)
Phosphoglucomutase reacton?
Converts glucose 1-P to glucose 6-P
- At the end of glycogen phosphorylase’s action, where only G-1-P remains
- Also 1st step of glycogen synthesis
- reversible reaction
Glucose-6-phosphate translocase action?
Enzyme that transports G-6-P into Endoplasmic Reticulum on LIVER/kidney cells
- for release to body after broken down to free glucose
Note: in muscle G-6-P is kept in cells for its own use
Reaction catalyzed by UDP-glucose phosphorylase?
Requirement?
Glucose 1-P –> UDP-glucose +PPi; 2nd reaction in glycogen synthesis
UTP is required
Also PPi breaks down to 2Pi
Reaction catalyzed by glycogen synthase?
Requirement?
UDP-glucose + Glycogen(n residues) –> Glycogen(n+1 residues) + UDP
One unit at a time
Requirement: Needs a “glycogen primer” of at least 4 residues
Reaction catalyzed by glycogenin?
Forms primer for glycogen synthesis
- UDP-glucose + tyrosine on glycogenin –> glucose-o-tyrosine benzyl on glycogenin + UDP
Makes 8mer primer, residues added on non-reducing end
Reaction catalyzed by (1,4–>1,6)- transferase?
- Branch formation on glycogen
- removes 6-8mer from non-reducing end, attaches it via 1,6 alpha linages, four residues from branch point
Glycolysis regulation actions for:
- Glucose 6 phosphate
- ATP
- AMP
- Glucose
- Glucose 6 phosphate: inhibits glycogen phosphorylase, activates glycogen synthase
- ATP: inhibits glycogen phosphorylase
- AMP: activate glycogen phosphorylase (MUSCLE ONLY; Ca2+ also activates glycogen breakdown via nerve stimulation only)
- Glucose activate glycogen phosphorylase (LIVER ONLY)
Important enzymes in fructose metabolism
- Fructokinase: requires ATP, Fructose to Fructose 1-6-P
- Aldolase B: Fructose 1-6-P to DHAP and Glyceraldehyde
(Cause of hereditary fructose intolerance)
Eventually gets converted to G3P, used in glycolysis
Important enzymes in sorbitol metabolism
- Aldose reductase: Needs NADPH; Glucose to Sorbitol (alcohol of fructose), produced when too much glucose
- Sorbitol dehydrogenase: Need NAD+, Converts sorbitol to fructose
- Often sorbitol accumulation is due to hyperglycemia
Important enzymes in galactose metabolism
- Galactokinase: ATP needed, Galactose to galactose-6-P
- Galactokinase deficiency leads to increase galactose in blood and urine, galacticol accumulation, results in cataracts
- Galactose 1-P Uridyl transferase: Needs UDP galactose (from UDP Glucose via epimerase); converts Galactose 1 P to Glucose 1 P
- Deficiency results in classic galactosemia, can cause liver damage and other issues
Then converted to glucose 6 P (phosphoglucomutase) for glycolysis/Glucose
Glycogen metabolism via Insulin (mechanism)
in liver and muscle
- Stimulates cAMP phosphodiesterase
- to decrease cAMP levels
- reducing PKA activity
- Increases hepatic protein phosphatase activity
- this inhibits glycogen phosphorylase and its activator, phosphorylase kinase
- stimulates glycogen synthase (via dephosphorylation) to increase glycogenesis
Glycogen metabolism via Glucagon (mechanism)
only in liver
- stimulates G-protein
- this stimulates adenylyl cyclase
- increases cAMP
- increases PKA activity
- PKA phosphorylates and activates glycogen phosphorylase and phosphorylase kinase (its activator)
Glycogen metabolism via Epinephrine (mechanism)
- PKA path in muscle only, both PKA and PLC in liver
- epi stimulates g-protein
- G protein stimulates PKA
- phosphorylation of glycogen phosphorylase and phosphoryl kinase (its activator)
- epi also stimulates phospholipase C
- PLC splits PIP2 to IP3 and DAG
- DAG activates protein kinase C directly, inactivates glycogen synthase (via phosphorylation)
- IP3 stimulates Ca2+ release to ER, which also activates PKC (which inactivates glycogen synthase)
- Ca2+ also binds to calmodulin to activate calmodulin dependent kinase and phosphorylase kinase (which activates glycogen phosphorylase)
- Calmodulin dependent kinase and phosphoryl kinase inactivate glycogen synthase
Lipase (FA breakdown)
- TAG to glycerol and fatty acids
- Regulated by epinephrine and glucagon (because they activate PKA which activates hormone-sensitive lipase in the liver)
- Insulin activates protein phosphatase 1 which inactivates HSL
*Note: FA are carried in blood via albumin to rest of thebody
Thiokinase
- Converts FA to Fatty acyl CoA
- Requires ATP
Rate limiting step in fatty acid breakdown?
Carnitine shuffle in long chain fatty acid oxidation
- In intermembrane space of mitochondria:
Attaches Carnitine to FA CoA via CPT-1 (carnitine palitoyl transferase 1) to facilitate diffusion across inner mitochondiral membrane
**CPT 1 is inhibited by Malonyl CoA (product during synthesis, Malonyl CoA in inhibited by high AMP and PKA)
- Once in Mit membrane, CPT-2 converts back to fatty acyl coA
Fatty Acyl coA synthetase
- ATP + FA to FAcyl AMP + PPi to Fatty Acyl coA
For intracellular activation of Fatty acyl coA
Acyl CoA Dehdrygeonases
Many Enzymes in reaction, convert Fattyl Acyl CoA to Enoyl CoA
- B oxidation reaction, FA breakdown
- Need FAD+, reaction produces FADH2
Enoyl CoA hydratase (general action)
Hydrolyses double bond in Enoyl CoA
3-hydroxyacetyl CoA dehydrogenase (general action)
Converts hydrated Enoyl coA (3-hydroxyacyl CoA) to ketone (3-ketoacyl coA)
-Need NAD+, to produce NADH
Beta-ketoacyl-CoA (general action)
- Breaks up 3-ketoacyl CoA to a fatty acyl CoA (-2 Cs) and acetyl coA
- The shorter FA COA is used for next round of beta oxidation (beta oxidation spiral)
- Need CoA
What happens when breaking down odd # C FA chain?
- Last 3 cs released as Propionyl CoA
- Converted to succinyl coA with three enzymes:
1. Propionyl CoA carboxylase (requires biotin, ATP, CO2)
2. Racemase- converts D to L isomer
3. Mutase- Converts to succinyl coA (needs vitamin B12)
HMG CoA Synthase
- EZ in ketone body synthesis
- Converts Acetoacetyl Co A to 3-hydroxy-3-methyl glutaryl CoA
** RATE LIMITING STEP
HMG CoA Lyase
Converts 3-hydroxy-3-methyl glutaryl CoA (3HMG CoA)
to AcetoAcetate
D-Beta hydroxybutrate dehydrogenase
- AcAc to D-Beta hydroxybutrate (BHB)
(NEED NADH) - AcAc also spontaneously releases acetone
Malic Enzyme
- Part of shuttle in FA synthesis to transport Acetyl CoA to cytosol from mitochondria (done by converting to citrate, then malate, then pyruvate)
- small amount of NADPH produced in step (nothing much)
- Converts Malate to pyruvate in cytosol
(PDH will then convert it back to Acetyl CoA)
Need NADP+
Acetyl CoA carboxylase
Converts Acetyl CoA to Malonyl CoA
Malonyl CoA can inhibit CPT-1 in carnitine shuttle in FA breakdown
***RATE LIMITING STEP IN FA SYNTHESIS
Needs Biotin
- Activated by citrate, insulin
- Inhibited by Palmitoyl CoA, AMP, Glucagon, Epinephrine
Fatty Acid Synthase
- 7 separate enzymes in complex that convert Malonyl CoA to Palmitate to Palmitoyl CoA!
- Uses CoA for 2 additional Cs at beginning
- EZ is a vitamin B derivative
- Panothenic acid is also part of complex (transfers intermediates between sites on EZ complex)
- Condensation, Decarboxylation, Reduction with NADPH x 2, dehydration reactions occur
**ELONGATIon beyond 16 C occurs in smooth ER, similar mechanism as FA synthase, Malonyl CoA and NADPH needed
Where does desaturation of FA occur? What enzyme?
- In ER, can only form chains with CIS double bonds
- Done by mixed function oxidase (desaturase)
- Need NADH, O2, Cytochrome b5
For trans double bonds, we need linoleate and linolenate
Regulation of FA synthesis
A. Fed state: INSULIN↑, Fatty acid synthesis active, malonyl CoA blocks fatty acid transport into mitochondria, preventing FA oxidation
B. Fasted state: GLUCAGON↑ , EPINEPHRINE↑ , INSULIN ↓; this activates Protein Kinase A via cAMP.
- Hormone sensitive lipase is active via phosphorylation by protein kinase A
- Acetyl CoA carboxylase is inactive (phosphorylated),
preventing malonyl CoA formation. - Lack of malonyl CoA permits fatty acids transport (CPT-I)
into mitochondria for oxidation.
