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  • Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,
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Proteins, Enzymes and Nucleic Acids A/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as sub Meiss , et al. 1999. JMB 288: 377–390

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Page 1: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Proteins, Enzymes and Nucleic Acids

The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substratesGregor Meiss, et al. 1999. JMB 288: 377–390

Page 2: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Proteins

Multipurposemolecules

Schematic representation of secondary structures:

Image from National Energy Research Scientific Computing Center

Page 3: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Proteins • Most structurally & functionally diverse group

• Function: involved in almost everything – enzymes (pepsin, DNA polymerase)– structure (keratin, collagen)– carriers & transport (hemoglobin, aquaporin)– cell communication

• signals (insulin & other protein hormones) • receptors

– defense (antibodies) – movement (actin & myosin)– storage (bean seed proteins)

Page 4: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Proteins• Structure

– monomer = amino acids• 20 different amino acids

– polymer = polypeptide• protein can be one or more polypeptide chains

folded & bonded together• large & complex molecules• complex 3-D shape

hemoglobin

H2O

Page 5: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Amino acids• Structure

– central carbon– amino group– carboxyl group (acid)– R group (side chain)

• variable group• different for each amino acid• confers unique chemical

properties to each amino acid– like 20 different letters of an

alphabet– can make many words (proteins)

—N—H

H C—OH

||O

R

|—C— |

H

Page 6: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Effect of different R groups:Nonpolar amino acids

Why are these nonpolar & hydrophobic?

nonpolar & hydrophobic

Page 7: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Effect of different R groups:Polar amino acids

polar or charged & hydrophilic

Why are these polar & hydrophillic?

Page 8: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Building proteins

• Peptide bonds– covalent bond between NH2 (amine) of one

amino acid & COOH (carboxyl) of another

peptidebond

dehydration synthesisH2O

Page 9: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Primary (1°) structure• Order of amino acids in chain

– amino acid sequence determined by gene (DNA)

– slight change in amino acid sequence can affect protein’s structure & its function• even just one amino acid change

can make all the difference!

lysozyme: enzyme in tears & mucus that kills bacteria

Page 10: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Sickle cell anemia

I’mhydrophilic!

But I’mhydrophobic!

Just 1out of 146amino acids!

Page 11: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Figure 5.21

PrimaryStructure

Secondaryand TertiaryStructures

QuaternaryStructure Function Red Blood

Cell Shape

subunit

subunit

Exposedhydrophobicregion

Molecules do notassociate with oneanother; each carriesoxygen.

Molecules crystallizeinto a fiber; capacityto carry oxygen isreduced.

Sickle-cellhemoglobin

Normalhemoglobin

10 m

10 m

Sic

kle-

cell

hem

og

lob

inN

orm

al h

emo

glo

bin

1

23

456

7

1

23

456

7

Page 12: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Secondary (2°) structure• “Local folding”

– folding along short sections of polypeptide– interactions between

adjacent amino acids• H bonds

weak bonds between oxygen and hydrogen atoms within the backbone.

– forms sections of 3-D structure• -helix• -pleated sheet

Page 13: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Tertiary (3°) structure• “Whole molecule folding”

– interactions between distant amino acids• hydrophobic interactions

– cytoplasm is water-based

– nonpolar amino acids cluster away from water

• H bonds & ionic bonds• disulfide bridges

– covalent bonds between sulfurs in sulfhydryls (S–H)

– anchors 3-D shape

Page 14: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Noncovalent Interactions between Proteins and

Other Molecules

Page 15: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Figure 5.19

Antibody protein Protein from flu virus

Page 16: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Quaternary (4°) structure• More than one polypeptide chain bonded together

– only then does polypeptide become functional protein

collagen = skin & tendons hemoglobin

Page 17: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Chaperonin proteins • Guide protein folding

– provide shelter for folding polypeptides– keep the new protein segregated from cytoplasmic

influences

Page 18: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,
Page 19: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Enzymes • Biological catalysts

– proteins (& RNA) – facilitate chemical reactions

• increase rate of reaction without being consumed• reduce activation energy• don’t change free energy (G) released or required

– required for most biological reactions– highly specific

• thousands of different enzymes in cells

– control reactionsof life

Page 20: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Figure 8.13

Course ofreactionwithoutenzyme

EA

withoutenzyme EA with

enzymeis lower

Course ofreactionwith enzyme

Reactants

Products

G is unaffectedby enzyme

Progress of the reaction

Fre

e en

erg

y

Page 21: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Induced fit model

– 3-D structure of enzyme fits substrate– substrate binding cause enzyme to change

shape leading to a tighter fit • “conformational change”• bring chemical groups in position to catalyze

reaction

Page 22: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

How does it work?

• Variety of mechanisms to lower activation energy & speed up reaction– synthesis

• active site orients substrates in correct position for reaction

– enzyme brings substrate closer together

– digestion• active site binds substrate & puts stress on

bonds that must be broken, making it easier to separate molecules

Page 23: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Enzymes and temperature• Different enzymes function in different

organisms in different environments

37°Ctemperature

reac

tion

rate

70°C

human enzymehot springbacteria enzyme

(158°F)

Page 24: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Rat

e o

f re

acti

on

0 1 2 3 4 5 6 7 8 9 10pH

(b) Optimal pH for two enzymes

Optimal pH for pepsin(stomachenzyme)

Optimal pH for trypsin(intestinal

enzyme)

Two enzymes function in the same organism, but in different environments

Page 25: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Compounds which regulate enzymes

• Inhibitors– molecules that reduce enzyme activity– competitive inhibition– noncompetitive inhibition– irreversible inhibition– feedback inhibition

Page 26: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Irreversible Inhibition

Page 27: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Reversible Inhibition

Page 28: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Reversible Inhibition

Page 29: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Regulatory site (one of four)

(a) Allosteric activators and inhibitors

Allosteric enzymewith four subunits

Active site(one of four)

Active formActivator

Stabilized active form

Oscillation

Nonfunctionalactive site Inactive form

InhibitorStabilized inactive form

Allosteric Regulation of Enzyme Activity

Page 30: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Active siteavailable

Isoleucineused up bycell

Feedbackinhibition

Active site ofenzyme 1 isno longer ableto catalyze theconversionof threonine tointermediate A;pathway isswitched off.

Isoleucinebinds toallostericsite.

Initial substrate(threonine)Threoninein active site

Enzyme 1(threoninedeaminase)

Intermediate A

Intermediate B

Intermediate C

Intermediate D

Enzyme 2

Enzyme 3

Enzyme 4

Enzyme 5

End product(isoleucine)

Feedback Inhibition of Metabolic Pathways

Page 31: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Nucleic AcidsInformationstorage

Page 32: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

proteins

DNA

Nucleic Acids• Function:

– genetic material• stores information

– genes– blueprint for building proteins

» DNA RNA proteins

• transfers information– blueprint for new cells– blueprint for next generation

Page 33: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Nucleotides

• 3 parts – nitrogen base (C-N ring)– pentose sugar (5C)

• ribose in RNA• deoxyribose in DNA

– phosphate (PO4) group

Page 34: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Types of nucleotides• 2 types of nucleotides

– different nitrogen bases – purines

• double ring N base • adenine (A)• guanine (G)

– pyrimidines• single ring N base • cytosine (C)• thymine (T)• uracil (U)

Purine = AGPure silver!

Page 35: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,
Page 36: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Nucleic polymer• Backbone

– sugar to PO4 bond

– phosphodiester bond• new base added to sugar of

previous base• polymer grows in one direction

– N bases hang off the sugar-phosphate backbone

Dangling bases?Why is this important?

Page 37: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Pairing of nucleotides• Nucleotides bond between

DNA strands– H bonds– purine :: pyrimidine– A :: T

• 2 H bonds

– G :: C• 3 H bonds

Page 38: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Copying DNA• Replication

– 2 strands of DNA helix are complementary

• have one, can build other

Page 39: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

When does a cell copy DNA?• When in the life of a cell does DNA have

to be copied?– cell reproduction

• mitosis

– gamete production• meiosis

Page 40: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Nucleic Acids Are Informational Macromolecules

• Genome—complete set of DNA in a living organism

Page 41: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

An interesting note…• ATP

Adenosine triphosphate

++

modified nucleotide adenine (AMP) + Pi + Pi

Page 42: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,
Page 43: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

RNA

Page 44: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Ghosts of Lectures Past

Page 45: Proteins, Enzymes and Nucleic Acids The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates Gregor Meiss,

Building proteins• Polypeptide chains have direction

– N-terminus = NH2 end

– C-terminus = COOH end– repeated sequence (N-C-C) is the

polypeptide backbone• can only grow in one direction


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