Enzyme Regulation Flashcards
regulation by reversible covalent modification
- alters enzyme activity by”
- causing a conformational change that affects catalysis
- altering cellular localization of the enzyme
- altering interactions with other proteins
- common reversible-phosphorylation, methylation, acetylation, glutathionylation, addition of ubiquitin
phosphorylation
-very common reversible covalent reactions modifications
glycogen phosphorylase
- regulated by reversible phosphorylation
- kinase activates enzyme
- phosphatase deactivates enzyme
regulation of irreversible covalent modification
-enzymes are synthesized in an inactive form and activated irreversibly in the time and/or place they are needed
zymogen
- inactive precursor form of a protease that is activated by a specific proteolytic cleavage by another protease
- digestive enzymes in stomach and pancreas-pepsin and chymotrypsin
- enzymes of clotting cascade
- caspases
digestive enzymes
- synthesized as zymogens and stored in pancreas
- activated upon release into the small intestine- and in response to hormones
- pancreas has tripsinogen, chymotripsinogen, and other zymogens in zymogen granules
- they are released to duodenum
- enteropeptidase cleave trypsinogen to trypsin, which cleaves all the others
irreversible enzyme activations in blood clotting
- maintenance of blood volume requires three rapid responses to injury
1. rapid activation of blood coag
2. localization of clot to injury
3. rapid termination after clot formation to prevent thrombosis - accomplished by series of sequential zymogen activations, most enzymes are serine proteases
clotting cascade
- each activated factor is a protease for the next factor
- factor VIIIa is a protein modulator missing in hemophiliacs
- potential for signal amplification at each step because each protease can cleave molecules then all those can cleave more
- thrombin in final-activates fibrinogen to fibrin for clot
- very confusing pathway and much communication between intrinsic and extrinsic
- need something to turn it off
- rapid activation of blood coagulation
- cascade of zymogen activations allows sequential activation of a series of serine proteases
- each activated protease can activate many target proteases, so signal is amplified
- regulation by irreversible covalent mod
- localization of clot to site of injury
- glutamate residues are modified to gamma-carboxyglutamate on several of the serine proteases of the cascade in a vitamin K dependent pathway
- calcium bridges the gamma carboxyglutamate (on serine protease) and membrane of site of injury
- regulation by reversible covalent mod and localization
pathway with vit K
-clotting protein —> (vit K dependent enzyme)–>
clotting protein with GLA modification
-can now bind via Ca to membrane that is injured
-anticoagulants (warfarin) block the enzyme and stop GLA modifications-leads to slow clotting
- rapid termination after clot formation to prevent thrombosis
- reverse zymogen activation cascade hydrolyzes clot
- irreversible covalent mod
opposing cascade
- plasminogen to plasmin via TPA (lose TPA-too many clots)
- fibrin stopped by plasmin and clot is hydrolyzed
anti-thrombin
- regulation by protein protein interaction
- serpin-binds at the active site of the protease and is cleaved and inhibits the enzyme
- inactivates thrombin and a number of other proteases of clotting cascade
- binds to thrombin and stops it with heparin
pancreatic trypsin inhibitor
- regulation by protein protein interaction
- inhibits inappropriately activated digestive proteases in pancreas
alpha1 antitrypsin
-inhibits elastase in lungs
anti-thrombin deficiency
- AD inheritance
- excessive clotting
- blood clots form inappropriately in legs or lungs
- observed after serious injury of during OC use
- can be fatal
- treated with long term anticoagulants
alpha1 antitrypsin deficiency
- too much active elastase in lung eventually causes COPD
- often diagnosed under conditions that recruit elastase producing macrophages to lungs-smoking, lung infection
- patient can’t inhibit elastase and experiences SOB
- sever deficiency often accompanied by liver disease
regulation by protein-protein interactions
- serpins are usually irreversible, but can have others
- signal transduction cascades are often regulated by this
- protein kinase A
- calmodulin-ca dependent interactions
protein kinase A regulation
- regulated by interaction of cAMP with regulatory subunits
- cAMP releases inhibition by regulatory subinits and activates catalysis
- cAMP turns on
clinical enzymology
enzymes as diagnostic tools
- diagnostic measurement of enzyme levels
- measurement of substrate of metabolite levels
- diagnosis of tissue damage or tumors by isozyme distribution
measurements of enzyme levels
- measure rate of product formation with time
- measurement of enzyme levels can be deduced from enzyme activity at saturated substrate-because should be at vmax
- at vmax, rate is proportional to amt of enzyme
- can measure product-rate of product appearance is rate of enzyme activity
- need excess substrate
coupled enzyme assays
- same principle, just need second step because can’t measure 1st product
- need to add substrates of first reaction, second substrate for second enzyme, and second enzyme in excess
use enzymes to measure metabolite or drug levels
- accurate, rapid, quantitation of a small molecule in a complex mixture such as serum or urine
- use and enzyme specific for substance to measure
- add large excess of enzyme, enough to convert substrate completely to product in short time
- measure amount of product formed, directly or indirectly
- speed, specificity, gentle conditions
- other factors in serum can interfere
-use coupled if needed to have detectable product
identification of tissue enzymes in serum
- non-plasma specific enzymes can appear in serum because of damage to tissue of origin or because of spillover due to overproduction in a tissue of origin
- can be detected by measuring level of enzyme activity, timing of appearance of activity, or presence of tissue specific isozymes
alanine aminotransferase
-viral hepatitis
amylase
acute pancreatitis
creatine kinase
muscle disorders and MI
lactate dehydrogenase isozyme 5
liver diseases
lipase
acute pancreatitis
phosphatase, acid
metastatic carcinoma of the prostate
phosphatase, alkaline
various bone disorders, obstructive liver diseases
enzymes and MI
CK, AspT, LDH
- appearance and timing are characteristic of MI
- need to measure at multiple times and look for cardiac isozymes
isozymes
- different forms of an enzyme that carry out the same reaction
- have different amino acid sequences, different chemical properties, and different enzymatic characteristics
- may have specific expression in different tissues or a specific pattern of expression during development
- can be distinguished by charge, monoclonal antibodies, differences in enzymatic properties or inhibitor sensitivities
LDH and CK isozymes
- in heard LDH1, in muscle LDH5
- CK MB in heart only
- CK3 in skeletal and cardiac muscle
isozymes can be distinguished by
- electrophoretic properties
- isoform specific monoclonal antibodies
- enzymatic properties (Km, Vmax, substrate preference)
- inhibitor sensitivity
- other characteristics
