Lecture 11-15 Flashcards
Chemical rxn rate
Always expressed in M/sec
Rate constant
Has units that allow rxn rate to have units on M/sec E + S EX E + P k1 = M-1 x sec-1 k2 = sec-1 k3 = sec-1
Michaelis-Menten assumptions
- [S]tot»_space; [E]tot such that [S]tot = ([S]free + [EX]) = [S]free
- Conservation of enzyme such that [E]tot = [E]free + [EX]
- [P] = 0 throughout measurements such that EX -> E + P is unidirectional
- Enzyme remains fully active throughout measurements
Steady state assumption
d[EX]/dt = 0. Constant flow through each step
v
Initial rate or velocity. v = k3[EX]
Vmax
Maximal rate or velocity. Vmax = k3[E]tot
Michaelis-Menten equation
v = (Vmax)/(1 + Km/[S])
Km
Kinetic parameter that may or may not equal Kd. Km only equals Kd when k2»_space; k3. Gives substrate concentration for rate that is half of Vmax.
Km = (k2+k3)/k1
kcat
Turnover number. The number of product molecules formed by each enzyme active site per second. Frequency of catalysis. True constant that represents catalytic efficiency
Vm = kcat[E]tot
Kd
Thermodynamic parameter and measure of affinity. Lower Kd means higher affinity
Kd = k2/k1
Ordered ternary complex mechanism
Model of how enzymes use two substrates. A binds first and Q leaves last
E + A <> EA + B <> EAB <> EPQ <> EQ + P <> E + Q
Random ternary complex mechanism
Model of how enzymes use two substrates. No compulsory order for substrate addition or release. A or B could bind first and P or Q could leave first. Neither path alters EX and overall catalytic mechanism
Ping pong mechanism
Model of how enzymes use two substrates. Forms covalent rxn intermediate (E-X). Ex: chymotrypsin
E + A <> EA + P <> E-X + B <> E-XB <> E + Q
Stopped-flow apparatus
Designed to use both absorbance and fluorescence detectors to observe multiple rxn intermediates during pre-steady-state phase. Evaluates all rate constants and all “internal” equilibrium constants for a more complete picture of catalysis
Competitive inhibition
Reversible form of inhibition. Inhibitor binds in place of substrate at active site. Raising [S] can fully reverse inhibition. Poor drug model
Noncompetitive inhibition
Reversible form of inhibition. Inhibitor binds at separate site to substrate. Raising [S] cannot fully revers inhibition. Better model for effective drugs
Uncompetitive inhibition
Reversible form of inhibition. Substrate binding creates site for inhibitor binding. Rare inhibitory mode as transition from EX to E+P is fast
Metabolism
Totality of cellular processes that make and degrade chemical substances (metabolites), fueling and facilitating vital processes such as meiosis, locomotion, transport, genetics, evolution, etc
Anabolism
Pathways that synthesize biomolecules form simpler precursor metabolites
Catabolism
Pathways that degrade complex biomolecules yielding energy and/or forming simpler metabolites
Allosteric regulation
Reversible binding of regulatory molecules that alter enzyme conformation and activity (µsec-msec). Activators increase substrate binding/kcat. Inhibitors decrease substrate binding/kcat
Reversible covalent modification
Group from donor molecule is transferred to target enzyme to change catalytic activity and can be removed to reverse effects (msec-sec)
Induction/Repression
Concentration of enzyme is controlled at the gene and/or mRNA level (1-1000 sec)
Primary metabolites
Needed for normal operation of metabolic pathways and main cellular functions. Ex: AAs, nucleotides, RNA, DNA, B vitamins
Secondary metabolites
Those organic compounds not needed for cell growth, development, or reproduction. Many ward off pathogens/predators. Others protect against osmotic damage. Many are useful for treating illnesses. Ex: alkaloids, fungal metabolites
Pyruvate
Alpha-ketoacid of Ala
Oxaloacetate (OAA)
Alpha-ketoacid of Asp
Alpha-ketoglutarate
Alpha-ketoacid of Glu
Nutritionally essential AAs
Phe, Val, Trp, Thr, Ile, Met, His, Leu, Lys
Conditionally essential AAs
Arg essential for growth (childhood + pregnancy). Tyr essential when Phe is low. Cys essential when Met is low
Pepsin
Cleaves Phe, Leu, Glu
Chymotrypsin
Cleaves aromatic AAs
Trypsin
Cleaves Lys, Arg
Carboxypeptidase
Cleaves C-terminal AA
Elastase
Cleaves elastin (highly elastic protein in connective tissue)
Zymogen
Inactive form (precursor) of protease Pepsinogen --> Pepsin Chymotrypsinogen --> Chymotrypsin Trypsinogen --> Trypsin Procarboxypeptidase --> Carboxypeptidase
Trypsinogen activation
Stored in secretory vesicles with trypsin inhibitor. Enterokinase (ectoprotein on intestinal mucosal wall) converts trypsinogen into trypsin
Chymotrypsinogen activation
Activated by trypsin (cleaves first) and by chymotrypsin (second cleavage)
Pepsinogen activation
Autocatalytic. Slow acid-catalyzed activation by stomach pH. As more pepsin accumulates, pepsin catalyzes activation of pepsinogen
Lysosomal/phagolysosomal pathway
Intracellular protein turnover pathway where lysosome uses acidic compartment to induce isoelectric expansion (partial unfolding). Low pH makes proteins more susceptible to proteolysis
Ubiquitin-dependent pathway
Intracellular protein turnover pathway that enzymatically joins ubiquitin to poorly folded proteins. Ubiquitinated proteins are degraded in proteasomes
Proteasome
Barrel-like macromolecular protease complexes
Positive nitrogen balance
Intake > Excretion. Needed for growth (childhood and pregnancy), healing, convalescence
Negative nitrogen balance
Excretion > Intake. Occurs during starvation, malnutrition, disease, injury
Marasmus
Malnutrition associated with extensive tissue and muscle wasting. Little/no edema. “Protein-energy malfunction” resulting from inadequate intake of protein and calories. Severe deficiency in nearly all nutrients
Kwashiorkor
Acute childhood protein malnutrition. Inadequate protein intake but normal caloric intake. Characterized by irritability, enlarged liver, abdominal edema caused by hypoalbuminemia
Transamination exceptions
Pro, Hyp = have secondary amines that cannot undergo transamination
Lys = would cyclize to form toxic nonmetabolite
Thr = would dimerize to form toxic nonmetabolite
Glutamate Dehydrogenase (GDH)
Major route for oxidative deamination. Regenerates a-ketoglutarate and provides ammonia. GDH located in mitochondrial matrix. Couples with transaminases. Uses NAD+ to drive deamination. Uses NADPH to drive amination
Glu + NAD+ + H2O <> a-KG + NADH + NH3
Glutaminase
Catalyzes hydrolysis of glutamine. Widely distributed in mitochondria to avoid futile cycle with glutamine synthetase
Gln + H2O <> Glu + NH3
Asparaginase
Catalyses hydrolysis of asparagine
Asn + H2O <> Asp + NH3
Histindinase
Catalyzes deamination of histidine
His <> Urocanate + NH3
Glutamine synthetase
Main way to trap NH3. Formation of Gln provides major inter-organ nitrogen shuttle to avoid direct transfer of NH3. Gln is a major source of nitrogen for many biosynthetic rxns. Uses gamma-glutamyl-P as essential intermediate
Glu + ATP + NH3 <> Gln + Pi + ADP + H+
Carbamoyl-Phosphate Synthetase I (CPS-I)
Main ammonia-assimilating rxn in mitochondria. Highly energy dependent. First step resembles glutamine synthetase.
2 ATP + HCO3- + NH3 <> 2 ADP + HPO4(2-) + Carbamoyl Phosphate
Location: mitochondria Substrate: ammonia Affinity for NH3: high Affinity for Gln: none Pathway: urea cycle Activator: N-acetyl-glutamate
Carbamoyl-Phosphate Synthetase II (CPS-II)
Uses transfer tunnel to move unprotonated NH3 from glutamine-hydrolysis site to biosynthetic site. Essential -SH group generates a gamma-glutamyl thioester that occupies active site - permitting unprotonated NH3 transfer.
Location: cytosol Substrate: glutamine Affinity for NH3: none Affinity for Gln: high Pathway: pyrimidine nucleotide biosynthesis
