biochem unit 4

what are the mechanisms of catalysis 

• covalent catalysis 

• acid base catalysis 

• low barrier hydrogen bonds

• metal ion catalysis

biochemical nucleophile serine

-OH → found in serine proteases (chymotrypsin, trypsin)

cysteine as a biochemical nucleophile

-SH → found in cysteine proteases (cascades, papain)

lysine as a biochemical nucleophile

-NH2 → important in schiff base formation (aldose)

histidine as a biochemical nucleophile

(imidazole group) → can act as a nucleophile and base 

water as a biochemical nucleophile

acts as a nucleophile in hydrolysis reactions

in covalent catalysis

the enzyme forms a temporary covalent bond with the substrate creating a reactive intermediate the facilitates the reaction

chymotrypsin 

a protease that uses both covalent and acid/base catalysis 

covalent catalysis increases the rate of biochemical reactions by

stabilizing high energy intermediates via covalent bonds

chymotrypsin cleaves the carboxyl side of large hydrophobic aromatic amino acids such as

phe, met, tyr and trp

acid-base catalysis 

a proton is transferred to catalyze reactions 

specific acid base catalysis involves

H+ or OH- that diffuses into the catalytic center

in specific acid base catalysis the rate of the reaction on depends on

the pH of the solution, not the concentration of the buffer

in acid base catalysis the rate of the reactions determined by the concentration of

H+ for acid catalysis and OH for base catalysis 

“general” acid base catalysis

acids and bases other weak acids and bases

• the rate depends on ph and concentration of buffer

• all bronstead acids are h+ donors and bases are h+ acceptors and contribute to the rate

examples of enzymes that employ general acid-base catalysis

serine and aspartic acid proteases

in the active site of serine proteases, the serine residue is usually paired with a _____ to promote nucleophilic attack on the peptide bond

proton withdrawing group (histidine residue)

aspartyl promises activate a ____ to serve as the nucleophile rather than using a functional group on the enzyme itself

water molecule 

lower barrier hydrogen bonds

• special type of hydrogen bond where the proton is more equally shared between the donor and the acceptor rather than being strongly associated with one

• usually strong and play a critical role in enzyme catalysis by stabilizing transition states

the typical hydrogen bond strength is

10-30kj/mol

as distance between hetero atoms becomes smaller…..

H bonds become stronger 

name the type of hydrogen bonds on left and right

left = ordinary

right = low barrier hydrogen bond

describe the differences

Left: the hydrogen atom is between the two donor atoms - LBHB

Right: the H atom is covalently bound - ordinary

label the types of hydrogen bonds 

A= weak

B/C= low barrier

As the distance between the hydrogen bonds gets shorter,,,

the highest bond energy is observed

the steps for low barrier hydrogen bonds

1. S binds to E via hydrogen bonding

2. 2. hydrogen bond strengthens/shortened and transition state stabilizes

3. transition state collapses through cleavage or bond formation

4. LBHB reverts to normal hydrogen bond

common residues for hydrogen bonding 

Asp, Glu, His and Tyr 

_____ in an enzymes cofactors interact with the substrate stabilizing the transition state and orienting the substrate for reaction

metal ions

mechanistic steps for metal ion catalysis

water activates, substrate binds, transition state is stabilized, leaving group leaves

three ways enzymes are regulated

• presence of allosteric regulators or inhibitors

• genetic regulation

• compartmentation

the adenylyl cyclase reaction

• converts ATP to cyclic AMP, a second messenger involved in signal transduction.

• driven forward by hydrolysis of pyrophosphate by the enzyme inorganic pyrophosphatase

cAMP

• intracellular agent of extracellular hormones

• second messanger

• hormone binding stimulates GaBy protein binding to GTP triggering its subunit

binding of Ga(GTP) to ______ activates cAMP production 

adenylyl cyclase

hormone binding to its receptor leads via _____ activation to cAMP synthesis

G-protein

cyclic AMP- dependent protein kinase is composed of

catalytic and regulatory subunits

Cyclic AMP-depdent protein kinase is a ______ in mammalian cells 

170-LD R2C2 tetramer 

in cyclic AMP the two regulatory subunits bind ___ equivalents of cAMP each; and then cAMP binding releases the R subunits from the Catalytic subunits

two

in cAMP dependent protein kinase the catalytic subunits are enzymatically active as ___

monomeric forms

genetic regulation of enzyme levels 

• transcriptional control, gene expression can be upregulated or down regulated in response to needs 

example of genetic regulation of enzyme levels

lac operon in prokaryotes

• presence of lactose upregulates the expression of lacZ, lacY, lacA

• absence of lactose down regulates gene expression

enzyme regulation by compartmentation

• enzymes and regulatory molecules are in separate compartments so that opposing pathways are either close together or far apart to increase efficiency

enzymes may also be attached to

cytoskeleton or a membrane

definition of metabolism

the sum of all chemical reactions in living organisms

the two types of metabolism 

catabolism and anabolism 

catabolism

breaking down molecules to produce cellular energy

anabolism

building biomolecules using cellular energy

glycolysis is catabolic or anabolic

catabolic

glycogenesis catabolic or anabolic

anabolic

phototrophs metabolic pathways

• use CO2 or organic matter and light to produce cellular energy

heterotrophs or chemotrophs 

• use CO2 or organic matter and REDOX active molecules to produce cellular energy 

autotrophs vs heterotrophs

• autotrophs - use CO2

• heterotrophs - use organic carbon

the three stages of catabolism

• large biomolecules are broken down into building blocks and then degraded into common product and then simple end products

what are the common degradation products of catabolism 

pyruvate and acetyl coa 

what comes before the citric acid cycle

acetyl coa

what comes before acetyl coA

pyruvate, fatty acids other than amino acids 

what comes after glucose but before pyruvate for polysaccharides

glyceraldehyde 3 phosphate (glycolysis)

end products in catabolism

h2o co2 and ammonia, atp

1- amino acids 

2- gluose

3-glycerol, fatty acids

4-pyruvate

5-acetyl-coa

6- citric acid cycle

7- oxidative phosphorylation 

parallel pathways of catabolism and anabolism must differ in at least _______ so that they can be regulated independently

one metabolic step

describe this image

• in A parallel sequences proceed by independent routes

• in B only one reaction has two different enzymes

ATP is considered _____ of the cell

the energy currency 

NADH and FADH2 are considered

electron carriers involved in redox reactions

what drives energonic reactions

reaction coupling of ATP

ATP is formed via ____ in photorophic cells

photosynthesis

energy requiring cellular activities are powered by

ATP hydrolysis that liberates ADP and Pi

NADH and FADH2 transfer electrons to the

electron transport chain

_____ collects electrons released in catabolism 

NAD+ 

ATP is formed by _____ in heterotrophic cells

catabolism

hydrogen and electrons released in catabolism are transferred as hydride ions to the _______ nucleotide

pyridine

in catabolism NAD forms NADH+ H+ IN ______ REACTIONS

dehydrogenase

in catabolism NADH passes ____ to other acceptors

H+

the final acceptor of electrons is

O2 and becomes reduced to h2o

the ultimate oxidizing agent

O2

atp hydrolysis is formed by

• biosynthesis

• osmotic work

• cell motility/muscle contraction

_____ is oxidative, _____ is reductive

catabolism, anabolism 

____ can be viewed as the carrier of electrons from catabolic reactions to anabolic

NADPH

NADPH can be described also as

reduced nicotinamide adenine dinucleotide phosphate

biosynthesis usually relies on 

reducing equivalents from NADPH

proteins, carbohydrates and lipids are good sources of chemical energy because

their carbon is reduced

the oxidative reactions of catabolism release reducing equivalents from carbohydrates, lipids and proteins often in the form of

hydride ions

chains of -ch2- groups are the most ____ form of reduced carbon 

energy rich source of chemical energy.

______ is the final product of catabolism and the most oxidized form of carbon

carbon dioxide

____ is an example of catabolism

glycolysis

the citric acid cycle and oxidative phosphorylation take place in 

the mitochondria 

glycolysis occurs in

the cytosol

glycolysis turns one glucose molecule into

2 pyruvate during cellular respiration.