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Flashcards in ch 10 Deck (108):
1

4 basic regulatory strategies

1. allosteric control
2. multiple forms of enzymes (isozymes)
3. reversible covalent modification
4. proteolytic activation

2

what is allosteric control

proteins contain distinct regulatory sites and multiple functional sites. binding of regulatory molecules triggers conformational changes that affect active sites. display cooperativity

3

what are information transducers

signal changes activity or information shared by sites

4

how do multiple forms of enzymes allow for regulation?

different isozymes used at distinct locations or times. differ slightly in structure, km, and vmax values, and in regulatory properties

5

how does reversible covalent modification work?

activities altered by covalent attachment of modifying group, mostly a phosphoryl group

6

how does proteolytic activation allow for regulation

irreversible conversion of an inactive form (zymogen) to an active enzyme

7

what type of regulatory mechanism does aspartate transcarbamoylase use?

allosterically inhibited by the end product of its pathway

8

what type of mechanism does hemoglobin use?

binds O2 cooperatively

9

what kind of regulation are isozymes specific to?

regulation specific to distinct tissues and developmental strategies

10

what type of regulation does chymotrypsin use?

activated by proteolytic cleavage

11

what does aspartate transcarbamoylase catalyize?

first step in the biosyntehsis of pyrimidines

12

what is ATCase inhibited by?

feedback inhibition: inhibited by CTP final product of the ATCase controlled pathway

13

Where does CTP bind?

CTP is structurally different form substrates of the reaction and thus binds to allosteric sites distinct from active site (allosteric inhibitor).

14

are the active site and regulatory sites on ATCase on the same or on different polypeptide chains

(in ATCase specifically, catalytic sites and regulatory sites are on different polypeptide chains)

15

how do you separate ATCase into regulatory and catalytic (r and c) subunits?

addition of p-hydroxymercuribenzoate, which reacts with cysteine residues, followed by ultracentrifugation to separate the subunits by size

16

which of the 2 subunits of ATCase is the regulatory subunit/catalytic subunit?

the larger subunit (5.85) is the catalytic subunit, and the smaller subunit 2.85 is the catalytic subunit

17

describe behavior and structure of catalytic subunit of ATCase

has catalytic activity, does not respond to CTP, does not exhibit sigmoidal behavior. (has 3 chains 34 kDA each)

18

describe behavior and structure of regulatory subunit of ATCase

can bind CTP but no catalytic activity. has 2 chains 17kda each.

19

what happens when regulatory and catalytic subunits of ATCase are combined

combine rapidly to yield 2 catalytic trimers and 3 regulatory dimers in the full complex

20

describe ATCase structure

2 catalytic trimers are stacked on one top of the other, linked by 3 dimers of the regulatory chains. each r-chain within a regulatory dimer interact with a c-chain within a catalytic trimer through a structural domain stabilized by a zinc ion bound to four cysteine residues

21

how does p-hydroxymercuribenzoate dissociate catalytic and regulatory subunits?

mercury binds strongly to the cysteine residues displacing zinc and destabilizing this domain

22

what is PALA

PALA is a bisubstrate analog (analog of the 2 substrates) that resembles an intermediate along the pathway of catalysis - it is a potent competitive inhibitor of ATCase; it binds to and blocks active sites.

23

how was PALA used to locate active sites of ATCase?

the enzyme was crystallized in the presence of PALA

24

where does PALA bind?

PALA binds at sites lying at the bounadries between pairs of c chains within a catalytic trimer

25

describe the active site of ATCase

most of the residues belong to one subunit, but several key residues belong to a neighboring subunit - because the active sites are at the subunit interface, each catalytic trimer contribues 3 active sites to the complete enzyme

26

how does the ATCase-PALA complex change as PALA binds?

enzynme expands. 2 catalytic trimers move from 12 angstroms apart and rotate approximately 10 degrees about their common threefold axis of symmetry. regulatory dimers rotate 15 degrees to acommodate this motion

27

ATCase allostery

presence or absence of susbtrate determines state: 2 forms in equilibrium T and R state. T state has lower affinity for substrates and lower catalytic activity.

28

does ATCase follow the concerted mechanism or the sequentional model

concerted model because it follows an all or none change in enzyme shape

29

describe equilibrium of T and R states of ATCase

Even in the absence of any substrate or regulators, ATCase exists in equilibrium between the R and T states

The T state is favored under these conditions by a factor of 200

30

why does having two different states create a sigmoidal curve?

r and t states have 2 different Kms (r state has a low Km and T state has a high Km) sigmoidal curve is the composite of the 2 michaelis menten curves

31

how does CTP inhibit ATCase

binding for CTP in each regulatory chain does not interact with catalytic subunit, but it changes the equilibrium toward the T state, decreasing net enzyme activity. enzyme becomes less responsive to the cooperative effects facilitated by substrate binding

32

how does ATP participate in regulation of ATCase. what happens when atp concentration is high

ATP is an allosteric regulator that increases reaction rate at a given aspartate concentration. At high concentrations of ATP, kinetic profile shows less pronounced sigmoidal behavior

33

explain the reasons for the effects of ATP on ATCase activity

1. high ATP concentrations indicates high purine concentration, increase in ATCase activity tries to balance purine and pyrimidine pools

2. high concentration of ATP indicates that there is significant energy stored in cell to promote mRNA synthesis and DNA replication--this leads to syntehsis of pyrimdines

34

what are isozymes

isozymes are enzymes that differ in amino acid sequence yet catalyze the same reaction

35

how do isozymes differ?

different kinetic paramters such as km or regulatory properties

eoncoded for by different genetic loci, which usually arise thorugh gene duplication and divergence

36

what does the existence of isozymes permit?

permits fine-tuning of metabolism to meet the particular needs of a given tissue or developmental stage

37

what is an example of isozymes?

lactase dehydrogenase--which functions in anaerobic glucose metabolism and glucose syntehsis

38

what are the 2 isozymic polypeptide chains for LDH? how similar are they?

H isozyme, highly expressed in the heart

M isozyme found in skeletal muscle.

75% identical

39

structure of functional LDH enzyme

tetrameric with many different combinations of the 2 subunits

40

how do rat heart LDH isozyme composition change?

rat heart LDH isozyme changes in the course of development. increase in H isozyme after birth

41

how does H4 isozyme and M4 isozyme differ?

H4 found in heart has a higher affinity for substrate and works best in the aerobic environment of the heart muscle

M4 has a lower affinity for substrate and works optimally in the anaerobic environment of the hard working skeletal msucle

42

what do different combinations of LDH result in?

they have different intermediate properties. the appearance of izoymes in the blood can be used as a diagnostic indicator of heart attack (increase in H4 relative to H3M means a heart attack has damaged heart muscle cells.

43

are covalent modifications reversible?

mostly

44

what are some irreversible covalent modifications?

Ras (GTPase) and Src kinase. modified with a lipid group that forces these enzymes to display
their active sites to the cytoplasmic side when they are attached to
the membrane


the attachment of ubiquitin: is
a signal that a protein is to be destroyed; eg. Cyclin, an important
protein in cell-cycle regulation, must be ubiquitinated and destroyed
before a cell can enter anaphase and proceed through the cell cycle

45

what are histones

histones are proteins that assist in packaging of DNA into chromosomes and also involved in gene regulation

46

why are histones acetylated and deacetylated?

more heavily acetylated histones are associated with genes that are being actively transcribed

47

how is histone acetylation regulated?

acetyltransferase and deacetylase enzymes are themselves regulated by phosphorylation

48

what is phosphorylation

phosphorylation is a highly effect means of regulating the activites of target proteins.

49

what is the most prevalent reversible covlalent modification

phosphorylation

50

what are kinases

kinases add phosphate group to proteins

51

how do kinases work

the terminal gama phosphoryl group of ATP is transferred to specific serine and threonine residues by one class of protein kinases and to specific tyrosine residues by another

52

what are phosphatases

protein phosphatases reverse the effects of kinases by catalyzing the hydrolytic removal of phosphoryl groups attached to proteins

53

what happens when an enzyme is dephosphorylated (structurally)

unmodified hydroxyl containing side chain is regenerated and orthophosphate (pi) is produced

54

why are phosphorylation and dephosphorylation not considered the reverse of each other?

each is irreversible under physiological conditions. no enzyme does both reactions

55

what does the rate of cycling between phosphorylated and dephosphorlyated states depend on?

relative activities of kinases and phosphatases

56

signal for phosphorylase kinase or glycogen synthase kinase 2

ca2+ and calmodulin

57

signal for protein kinase c

diacylglycerol

58

signal for target-specific enzymes, such as pyruvate dehyrogenase kinase and branched-chain ketoacid dehydrogenase kinase

metabolic intermediates and other local effectors

59

how does adding a phosphoryl group contribute to great alterations of protein structure?

1. a phosphoryl group adds 2 negative charges to a modified protein
- electrostatic interactions in the unmodified protein can be disrupted and new electrostatic interactions can be formed, altering the structure of the proteins


60

phosphate group can form 3 or more ___________

hydrogen bonds

61

phosphorylation can change conformational equlibrium between ____________

different functional states

62

how is phosphorylation used to meet timing needs of physiological processes?

phosphorylation and dephosphorylation can take place in less than a second or over a span of hours

63

how does phosphorylation result in signal amplification?

a single kinase can phosphorylate hundreds of target proteins ina short interval, these target proteins may also further participate in the signal cascade

64

how is energy status of a cell linked to cell metabolsim

ATP is cellular energy currency, and using it as a phosphoryl group donor links energy status to regulation of metabolism

65

how is a kinase activated?

multi-step process initiated by hormones which trigger formation of cyclic amp (cyclization of atp)

66

what is cyclic amp

intracellular messenger in mediating the physiological actions of hormones

67

what is cAMP usually activated by?

protein kinase A (pka)

68

what does PKA do?

alters activites of target proteins by phosphorylating specific serine or threonine residues.

69

what is the structure of PKA in muscle?

it has 2 subunits 1 49 kd regulatory subunit with a high affinity for cAMP, and 1 38 kd catalytic subunit

70

how does pka behave in the absense and presence of cAMP

in the absense of cAMP, regulatory catalytic subunits form R2C2 complex. binding of 2 molecules of cAMP to each of the regulatory subunits leads to dissociation of R2C2 into R2 and 2 C2 subunits (free catalytic subunits are then enzymatically active)

71

how does binding of cAMP activate the kinase

every R chain contains a pseudosubstrate sequence that occupies the catalytic site of C preventing entry of protein substrates. binding of cAMP to the R chains allosterically moves pseudosubstrate sequences out of the catalytic sites. released c chains are then free to bind and phosphorylate substrate proteins

72

describe the active site cleft of pka

-the catalytic subnit of pka contains a bound 20 residue peptide inhibitor.
-atp and part of the inhibitor fill a deep cleft between the lobes.
-the smaller lobe makes many contacts with ATP-mg2+,
-the larger lobe binds peptide and contirbutes to key catalytic residues.
-2 lobes move closer together upon susbtrate binding.

73

why pka structure have a broad significance?

residues 40-280 constitue a conserved catalytic core that is common to essentially all known protein kinases

74

how is the pseudosubstrate sequence different from the natural consensus? how does it bind?

inhibitor peptide: arg-asn-ala-ile
it replaces serine with alanine. the 2 arginine side chains of the pseudosubstrate form slat bridges with 3 glutamate carboxylases, isoleucine fits in a hydrophobic groove formbed b 2 leucine residues in enzyme

75

some enzymes are syntehsized as inactive precursors that are activated by cleavage. what is the inactive precursor called?

a zymogen or pro enzyme

76

is ATP needed for cleavage?

no

77

how does proteolytic activation contrast with llosteric control/reversible covalent modification?

it occurs just 1 time in the life of an enzyme molecule

78

how is proteolytic activation used for digestive enzymes?

The digestive enzymes that hydrolyze proteins are synthesized as
zymogens in the stomach and pancreas

79

how is blood clotting mediated by proteolytic activation?

Blood clotting is mediated by a cascade of proteolytic activations
that ensures a rapid and amplified response to trauma

80

how is insulin mediated by proteolytic activation

Some protein hormones are synthesized as inactive precursors
-for example, insulin is derived from proinsulin by proteolytic
removal of a peptide

81

how is collagen derived from proteolytic activation

The fibrous protein collagen, the major constituent of skin and bone,
is derived from procollagen, a soluble precursor

82

what developmental processes are controlled through proteolytic activation?

Metamorphosis of a tadpole into a frog, large amounts of
collagen are resorbed from the tail in the course of a few days.
Likewise, much collagen is broken down in a mammalian uterus after
delivery. The conversion of procollagenase into collagenase, the
active protease, is precisely timed in these remodeling processes.

83

how is apoptosis mediated by proteolytic activation

Programmed cell death, or apoptosis, is mediated by proteolytic
enzymes called caspases, which are synthesized in precursor form as
procaspases
-when activated by various signals, caspases function to cause cell
death in most organisms
-apoptosis provides a means sculpting the shapes of body parts in
the course of development

84

zymogens of pepsin, chymotrypsin, trypsin, carboxypepdiase, and elastase

pepsinogen, chymotrypsinogen, trypsinogen, procarboxypeptidase, proelastase

85

where in the pancreas are enzymes and zymogens synthesized and stored?

acinar cells of the pancreas, and stored in the membrane bound granules. zymogen granules accumulate at the apex of teh acinar cell

86

how does the pancreas release enzymes?

when cell is stimulated by a hormonal signal or nerve impuse, the contens of the granules are released into a duct leading to duodenum

87

what does chymotrypsin do?

hydrolyzes proteins in the small intestine

88

how is chymotrypsinogen coverted into a fully active enzyme?

chymotrypsinogen is cleaved between arginine and isoleucine by trypsin, resulting in pi-chymotrypsin which acts on other pi chymotrypsin molecules

2 dipeptides are removed to yield alpha chymotrypsin, the stable form of the enzyme. 3 resulting chains in alpha chymotrypsin remain linked to one another by 2 interchain disulfide bonds

89

what conformational changes occur when chymotrypsinogen is cleaved?

amino terminal group of isoleucine turns inward to form an ionic bond with aspartate 194 in the interior of chymotrypsin molecule

90

what stabilizes the chymotrypsin transition state during catalysis?

hydrogen bonds between the negatively charged
carbonyl oxygen atom of the substrate and two NH groups of the
main chain of the enzyme; one of these NH groups is not
appropriately located in chymotrypsinogen, and so the oxyanion hole
is incomplete

91

what is the master activation step for the digestion of proteins? and describ the cascade of events

eteropeptidase (master activation step) hydrolyzes a lysine-isoleucine peptide bond to convert trypsinogen to trypsin. trypsin is the common activator of all pancreatic zymogens

92

what is special about protease inhibitor, pancreatic trypsin inhibitor?

exibit extremely tight binding to active site. (inhibits trypsin)

93

why does pancreatic trypsin inhibitor bind so tightly?

pancreatic trypsin inhibitor is a very effective substrate analog, multiple hydrogen bonding interactions, highly complementary to the enzymes active site

94

why does pancreatic trypsin inhibitor exist?

trypsin molecules activated in the pancreas or pancreatic ducts could severely damage those tissues leaving ro acute pancreatitis. tissue necrosis may result from the premature activation of proteolytic enzymes

95

what are the 2 clotting pathways?

intrinsic clotting pathway
extrinsic clotting pathway

96

what is the intrinsic clotting pathway

activated by exposure to anionic surfaces on rupture of the endothelial lining of the blood vessels, surfaces serve as binding sites for factors in the clotting cascade

97

what is the extrinsic clotting pathway

triggered by substances released from tissues as a result of trauma to them.

98

what ensures fast clotting response

very small amounts of initial factors suffice to trigger the cascade

99

why are both intrinsic and extrinsic clotting necessary ?

both pathways converge on a common sequence of final steps to form a clot composed of the protein fibrin

100

what is the final step i nthe clotting cascade?

to convert fibrinogen into fibrin by thrombin

101

what is fibrinogen made of

3 globular units connected by 2 rods,

102

what does thrombin do?

cleaves 4 arginine-glycine peptide bonds in the central globular region of fibrinogen (removes a and b peptides) results in fibrin monomer

103

what do fibrin monomers do

spontaneously assemble into ordered fibrous arrays called fibrin

104

general steps of the formation of fibrin clot?

1. thrombin cleaves fibrinopeptides A and B from the central globule of fibrinogen
2. globular domains at the carboxyl terminal ends of the beta and gama chains interact with knobs exposed at the amino terminal ends of the b and a chain to form clots

105

how is a clot stabilized? what enzyme is used, how is it activated

by formation of amide bonds between side chains of lysine and glutamine residues in different monomers, cross linking reaction is catalyzed by transglutaminase which is activated from the protransglutaminase form by thrombin

106

what is hemophelia?

bleeding genetic disorder, failure to produce blood clots resulting in sustained bleeding. antihemophilic factor of the intrinsic pathway is missing or has reduced activity.

107

what is VIII (antihemophilic factor)

not a protease, but stimulates activation of factor x, the final protease of the intrinsic pathway

108

how were hemophiliacs treated in the past and how are they treated now?

used to be given transfusions of concentrated plasma containing factor VIII, now use recombinant DNA techniques to isolate factor VIII and express in cells grown in culture