Ch 10 Lecture 1

dots represent intersections

represents 500 or so reactions of metabolic pathways

pathways are enzymes

five hundred reactions

not all of them because we need regulation so that reactions that don't need to happen aren't taking place

allosteric control

isozymes

reversible covalent modification

proteolytic activation

enzyme present but in wrong conformation; interaction of enzyme with some other molecule, causing a change in shape so enzymes becomes active to inactive

evolutionarily speaking, some genes of enzymes have replicated many times--> multiple copies of genes within a genome

all are subject to mutation in different places, times, and rates

enzymes begin to diverge

They catalyze the same reaction, but use different specificities such as rates

they give cells ability to pick and choose the isozyme that functions best in the physiological environment

cells have a choice of which enzyme

• produces enzymes in an inactive (proenzyme) form
• enzymes synthesize but not in correct conformation to be active. Can be active in one or two ways, which is the reversible covalent mod and proteolytic enzymes

mostly through phosphorylation of serine, threonine, and tyrosine

family of kinases that phosphorylate serine and threonine. They pop off hydrogen and phosphate, altering side chain--> conformational change in enzymes, activating it

since it is reversible, another enzyme must take phosphate off (phosphatase- removes phosphate)

already produced and ready to go (switches on and off)

makes enzymes recyclable (no need to remake enzyme); saves energy

the most common way of activating enzyme inside of cell where ATP is readily available.

enzyme is present in proenzyme form, but, instead of adding something, a pep bond is cleaved, activating the enzyme

separate protease is required

happens outside of cells; one time deal; one protein is activated, you can't deactivate it.

its outside of the cell, you can't find it

you don't want random proteases to roam around inside cell

allosteric protein; first step in a pathway leading to production of nucleotide CTP; activated when we need CTP

• two subunits
• 1) amino acid aspartate
• 2) carbamoyl phosphate

both can be used outside this reaction as well

it is a regulation of ATCase

• if there is enough CTP in the syste, the end product, CTP, inhibits ATCase to stop production
• Due to this, ATCase is the commiteted step. Once it catalyzes that reaction, it goes on to produce it. If not needed, hydrogen needs to be inhibited at this reaction.

is not the product of the reaction. It is the product of the pathway that has several reactions.

CTP production is a multistep reaction pathway

• has T and R state; has two catalytic triads
• self-interaction in R state versus other interactions in the T state

With every substrate constant, we vary the concentration of inhibitor. The more inhibitor that is added, the less the reaction proceeds, showing that CTP inhibits ATCase

No CTP is present. In a cuvette, buffer held constand. We change [Aspartate]. 

ATCase is an allosteric enzyme. 

This graph shows the rate increases as the amount of substrate increases, eventually leveling off

• it has multiple subunits being held by S-S- bonds
• ``

reagent broke the SS bonds and bonds to cysteine residues. They took them and ran it through cesium chloride. 

Large centrifuge tube with layer of proteins on top. Let it spin overnight and at a higher speed. Different proteins willm igrate down and rest. Larger moves further down. Wherever a band forms shows what the mass is

They then assayed it

12 subunits to begin with in the very begining before breaking it down

after breaking a bond, you get 2 peaks--> one representing a dimer (r) and another representing a trimer (C)

It has three subnits

Different peaks corerspond to two different types of subunits

regulate activity o eznymes and come in pairs

the trimers are held together by the dimers

• a reaction intermediate
• it is not quick, but it is quick enough to prevent us from freezing

to study, scientists creted an intermediate analog to study it. It can't have its phosphate group removed. Gets into active site and freezes ATCase in its active conformation--> X ray crystallography revealed structure when active versus inactive

active site

• side chains and interactions
• hold substrate in place

we have purified ATCase in tense state in cuvette--> add PALA--> binds to each subunit (6 active sites per ATCase= 6 reactions at one time)--> binds to active site--> T switches to R--> changes

space in middle gets longer. 12 angstrom separation

10 degree rotation relative to each other vertically

15 degree rotation horizontally

binds to regulatory subunits to prevent a conformational change

CTP helps maintain the T state

the subunits overlap and touch in the T state

pull apart and rotate

changes in one catalytic subunit, which is made of two lobes with hinge in the middle

• T state= side chains too far apart
• R state= movement of the two lobes; bring in closer proximity--> interaction

interactions across catalytic trimers allow it to happen

Specific amino acid side chains form ionic bonds over gap= holds T state stable and holds interactions across substrate

two lobes close and substrate fits in, allowing active site to be activated

interactions become intra (among) and hold the two lobes within

T state corresponds to low activity; not enough to keep the cell going and making CTP

concerted model: all six subunits switch at the same time. All are either T or all are R ; no intermediates

binds to help maintain the T state

• less active
• CTP binding
• more active
• substrate binding

lower activity in the presence of CTP

If you add ATP, it becomes more active. Cell is i a good energetic state,it can replicatei, needs nucleotides--> makes CTP