nucleic lec 6

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Questions and Answers

Which statement accurately describes the role of cofactors in enzymatic reactions?

Cofactors act as substrates during the reaction.

Cofactors can only provide energy for reactions.

Cofactors are not involved in the catalytic process.

Cofactors can carry activated chemical groups between enzymes. (correct)

What is the significance of the hydride ion in the reaction catalyzed by D-lactate dehydrogenase?

It facilitates the release of the substrate.

It is donated by the bound co-factor NADH. (correct)

It acts as a substrate for the enzyme.

It is a product of the reaction.

Which amino acid plays a critical role in shifting the pKa of histidine during the D-lactate dehydrogenase reaction?

Tyrosine

Cysteine

Arginine

Glutamate (correct)

How does D-lactate dehydrogenase ensure substrate specificity during its catalytic action?

Through favorable van der Waals interactions in a hydrophobic pocket. Signup and view all the answers

What role does the active site structure of D-lactate dehydrogenase play in the catalytic process?

It rotates to enclose the substrates and facilitate hydride transfer. Signup and view all the answers

Which of the following statements regarding the binding of pyruvate to D-lactate dehydrogenase is true?

Pyruvate stacks on the nicotinamide ring of NAD. Signup and view all the answers

Which elements are considered essential catalytic components in the D-lactate dehydrogenase family?

Arginine, histidine, and glutamate are essential for catalysis. Signup and view all the answers

What type of reaction occurs when D-lactate dehydrogenase reduces pyruvate?

Redox reaction Signup and view all the answers

Which of the following statements is true about the role of metal ions in enzymatic activity?

Metal ions can enhance the stability of enzyme-substrate complexes. Signup and view all the answers

How do hydrogen bonds contribute to the function of D-lactate dehydrogenase?

They stabilize the enzyme structure and assist substrate binding. Signup and view all the answers

What is the main function of carbonic anhydrases?

Hydrolyze CO2 Signup and view all the answers

How do D-LDH homologs differentiate their substrate specificity?

Through changes in their methyl binding pocket Signup and view all the answers

Which two residues are essential for the catalysis of carbonic anhydrases?

Cysteine and Histidine Signup and view all the answers

What determines an individual's blood type related to glycosyltransferases?

The specific version of glycosyltransferase inherited Signup and view all the answers

Which statement accurately reflects protein sequence divergence?

Different species may have proteins with slightly different sequences. Signup and view all the answers

Which amino acid change in D-LDH helps accommodate larger substrates?

Phe to Leu Signup and view all the answers

What role does zinc play in carbonic anhydrases?

It activates the water ion for reaction. Signup and view all the answers

What is the approximate sequence identity between the bacterial carbonic anhydrases and the pea enzyme?

11% Signup and view all the answers

How many essential residues are conserved in the active site between some carbonic anhydrases despite low sequence identity?

5 Signup and view all the answers

What kind of interactions might D-LDH have with its larger substrate due to structural changes?

Improved hydrophobic interactions Signup and view all the answers

What role do charged or titratable amino acids play in enzyme catalysis?

They are generally involved in catalytic mechanisms. Signup and view all the answers

How do peptides differ from globular proteins in terms of binding?

Peptides adapt their structure to the protein surface. Signup and view all the answers

What is one common feature of nucleic acid binding proteins?

They often utilize basic residues like arginine. Signup and view all the answers

Which type of chemistry is predominant in enzyme catalysis?

Acid-base and redox chemistry Signup and view all the answers

What is the significance of the active site structure in enzymes?

It organizes substrates for the catalytic reaction. Signup and view all the answers

Which of the following residues is commonly found in catalytic functions?

Histidine Signup and view all the answers

What type of protein interaction facilitates redox chemistry in enzymes?

External co-factors Signup and view all the answers

How do enzymes initiate the catalytic reaction?

By polarizing bonds and/or attacking reaction centers. Signup and view all the answers

What does the binding of peptides lead to in terms of conformational changes?

Loss of conformational entropy upon binding. Signup and view all the answers

What effect does the transient binding of Ran and RanGAP promote?

Exchange of GDP for GTP. Signup and view all the answers

What type of interaction do protein cofactors generally have with enzymes?

They can be tightly bound or covalently linked. Signup and view all the answers

What is the primary substrate that GTA transfers to the antigen?

N-acetyl glucosamine from UDP-GlcNAC Signup and view all the answers

Which substance is transferred to the antigen by GTB?

Glucose from UDP-Glc Signup and view all the answers

What are the two specific transfer processes described?

GTA transfers N-acetyl glucosamine and GTB transfers glucose. Signup and view all the answers

Which of the following correctly matches the enzyme with its substrate transfer?

GTA - UDP-GlcNAC Signup and view all the answers

What role does calmodulin play in response to calcium signals within a cell?

Calmodulin is a calcium-modulated protein that binds to target proteins in response to elevated calcium levels, regulating their activity. Signup and view all the answers

Describe the structural features of calmodulin that facilitate its function.

Calmodulin consists of two EF-hand motifs, each forming a four-helix bundle connected by a flexible linker. Signup and view all the answers

How do EF-hands contribute to calmodulin's ability to sense calcium levels?

EF-hands are helix-turn-helix motifs that are essential for binding calcium ions, thus enabling calmodulin to detect changes in calcium concentration. Signup and view all the answers

Identify the key residues involved in calcium binding within EF-hand motifs.

Two Asp residues and one Glu residue in the loop region are critical for calcium binding. Signup and view all the answers

What structural change occurs in calmodulin upon calcium binding?

Calcium binding alters the EF-hand helices from nearly parallel arrangements to right angles, unveiling a hydrophobic core. Signup and view all the answers

Explain how calmodulin regulates target proteins after binding to calcium.

The Ca-Calmodulin complex binds to non-polar regions of target proteins, using flexible methionines to adjust cavity shape for optimal interaction. Signup and view all the answers

Can you give an example of a protein regulated by calmodulin and describe its effect?

Calmodulin regulates calmodulin-dependent kinase, activating the kinase upon binding. Signup and view all the answers

What is the significance of the conserved residues in EF-hand motifs found in other calcium-binding proteins?

Conserved residues like Asp and Glu are vital for coordinating calcium ions, making these motifs effective across numerous calcium-binding proteins. Signup and view all the answers

Study Notes

Protein Function

Proteins have a vast array of functions in nature, arising from a combination of factors.
These factors include recognizing and binding other molecules (including proteins, nucleic acids, and small molecules).
They also catalyze chemical reactions, acting as enzymes.
Proteins can serve as substrates for modifications.
Combined with intrinsic structure and dynamics, these functions can result in complex roles.
Hemoglobin, for example, binds heme, reacts with CO₂, binds bisphosphoglycerate, and binds O₂, enabling oxygen transport throughout the body.

Binding

Cells contain numerous molecules like ions, proteins, nucleic acids, lipids, and metabolites.
These molecules constantly diffuse and collide within the cell (in bulk solution or within a membrane).
Individual proteins recognize specific molecules from this complex mixture.
Recognition implies a stable protein complex facilitating a biologically relevant effect.
These complexes form the basis for signaling events, catalysis, and protein localization.

Binding Energetics

Binding requires a lower energy state for the complex compared to isolated molecules.
Driving forces of binding are hydrogen bonds, van der Waals interactions, and hydrophobic interactions.
The energy required to displace water and loss of conformational heterogeneity contribute to a balanced energy equation.
Binding interaction generally maximizes the contact surface area.

Stable vs. Transient Interactions

Stable interactions (in a long period) exhibit micromolar or better affinity (even picomolar in certain cases).
Other processes require weak and fleeting interactions, typically within the micromolar to millimolar range, and a large fraction of the protein may remain unbound at any given time.

Small Molecule Binding

Cells contain diverse small (non-polymer) molecules like metabolites, building blocks, enzyme cofactors, and ions.
The binding site of a protein designed to recognize a specific small molecule is pre-organized to specifically bind that ligand.
This binding site is generally complementary to the ligand's properties.

Streptavidin

Streptavidin is a 159-amino acid protein from Streptomyces avidinii.
It acts as a biotin-scavenging protein (a crucial cofactor in carboxylation reactions like acetyl-CoA carboxylase in fatty acid synthesis).
Streptavidin exhibits exceptionally high affinity for biotin (~10^-14 mol/L).
It's widely used in biotechnology for reliably capturing biotin-tagged molecules, even at low concentrations.

Streptavidin Structure

Streptavidin consists of eight β-strands forming a beta sheet (up-down topology), with the first and last strands forming a beta barrel.
The functional tetramer originates from four streptavidin protomers in D₂ symmetry.

Streptavidin Binding Biotin

Biotin binds each protomer in the center of the beta barrel.
The apo structure's short helix rearranges into a loop to encapsulate biotin during binding.
During binding, typical structural transitions are observed.

Biotin Binding Details

Biotin's binding site is primarily non-polar.
Polar groups within the site are buried and stabilized by hydrogen bonds.
The hydrophobic effect contributes towards biotin binding.

Biotin Shape Complementarity

The streptavidin binding pocket precisely matches biotin's shape.
Nonpolar atoms are in close van der Waals contact.
Polar atoms are closer, forming shorter hydrogen bonds.

Protein-Protein Binding

Proteins can form both permanent and short-lived complexes.
Complexes often localize proteins within cells, modulate activity, or serve substrates/allosteric modulators for each other.
Protein-protein interactions typically involve extensive buried surface areas (minimum 700 Å²) for strong binding.
The interactions are usually similar to oligomeric interfaces, but potentially less hydrophobic if the complexes are temporary.

Ran and RanGAP Complex

Ran and RanGAP form a short-lived signaling complex.
Ran (purple) and RanGAP (grey) promote GDP-GTP exchange, leading to Ran's active site.
Individual proteins fold independently but transiently associate..
Proteins do not extensively reorient, the Ran active site only slightly restructures to promote GDP exchange.
Extended protein surfaces are buried between the proteins.

Ankyrin Complex in Erythrocytes

Ankyrin is a large helical repeat protein (in red).
It links the cytoskeleton to the cell membrane via proteins like spectrin.
In erythrocytes, it binds the regulatory domains of proteins like bicarbonate/chloride antiporter, Rh protein, and aquaporin.
The complex specifically anchors the cytoskeleton to the membrane.

Protein-Peptide Binding

Peptide binding differs from protein binding because peptides lack intrinsic structure.
This allows peptides to conform to the target protein surface.
Maximizing interactions comes at a cost, losing conformational entropy during binding.

Nucleic Acid Binding

Nucleic acid binding proteins mostly use multiple basic residues (arginine) to interact with the polyphosphate backbone.
Hydrogen bonds to exposed polar groups add specificity.
For RNA binding, stacking on exposed bases can contribute to stability.
Unstructured positive-charge-rich regions becoming ordered upon NA binding is common.

Enzyme Catalysis

Many proteins act as catalysts accelerating chemical reactions.
Most enzyme catalytic mechanisms are either acid-base chemistry (transferring protons), or redox chemistry (transfer of electrons or hydride ions).
Enzymes utilize multiple groups for acid-base chemistry, or external factors for redox chemistry.

Enzyme Active Sites

Enzymes bind substrates in conformations optimized for the reaction.
Catalytic residues, like polarizing bonds and attacking reaction centers, are nucleophilically or electrophilically, and/or remove or add protons or electrons.

Catalysis and Amino Acids

Catalysis heavily relies on a few charged or titratable amino acids.
Histidine is a commonly used catalytic residue even though not especially frequent.
Catalytic residues are typically charged or possess a pKa near neutrality.
Polar side chains are more frequently involved than nonpolar aliphatic side chains (like glycine, alanine, valine, isoleucine, leucine, proline).

Protein Cofactors

Enzymes and other proteins use diverse cofactors (e.g., pyridoxal phosphate, biotin, SAM).
Cofactors help perform chemically challenging tasks that amino acid side chains can’t.
Associated cofactors (tightly bound or covalently linked) remain permanently.
Cofactors can enable proteins to capture energy (light, e.g., chlorophyll) or utilise challenging groups (e.g., heme, biotin with CO2, cobalamin with CH3, FAD with H-, pyridoxal phosphate with NH3).
Cofactors can transport activated chemical groups between enzymes, like hydride (NADPH) and methyl (SAM).

D-Lactate Dehydrogenase

D-lactate dehydrogenase reduces pyruvate to D-lactic acid (redox reaction).
In bacteria, it is crucial in serine metabolism, as is a human-equivalent protein..
The active site is located in the cleft between two Rossmann domains.

DLDH Catalytic Groups

Proteins lack good hydride donors, so NADH provides hydride.
Proton transfer happens from histidine to pyruvyl groups; glutamate participates in histidine pKa shift.
Arginine is vital for polarising and activating the keto group.
These elements are essential and conserved amongst DLDH family in similar reactions.

D-LDH Domain Closure

D-LDH's small domain closure isolates substrates to ensure substrates cannot bind.

D-Lactate Dehydrogenase Binding

D-LDH binds a small substrate (pyruvate) enabling specificity by making favourable contacts with every atom.
Pyruvate stacks on the nicotinamide ring of NAD.
Catalytic residues contribute to binding.
Catalytic arginine and histidine form hydrogen bonds.

D-LDH Carboxylate Binding

Substrate carboxylate group is bound via hydrogen bonds from backbone and ligands.
Two backbone amide nitrogens bind the substrate carboxylate.
Tyrosine contributes to binding with a third hydrogen bond.

D-LDH Substrate Specificity

The methyl group within D-LDH is located in a hydrophobic pocket with Phe and Tyr.
Van der Waals interactions with other metabolites resemble pyruvate (but differe).
Phe and Tyr residues crucially maintain specificity.

Metal Ions

Many proteins bind specific metal ions for catalysis (e.g., metalloproteases), structure (e.g., zinc finger proteins), or regulation (e.g., calmodulin).
Metal ions are coordinated by conserved amino acids (Cys, His, Asp, or Glu; H₂O or S).
Transition metal ion-protein bonds are almost covalent.
Transition metals are fundamental participants in redox reactions.
Iron-sulfur clusters of proteins support electron transfer over longer distances.

Src Kinase ATP Binding

Src kinase binds ATP in a pre-organized pocket.
Hydrophobic residues in the pocket bind to adenine base + ribose.
Hydrogen bonds connect to backbone and help neutralize the PO₄ group .
A magnesium ion assists phosphate coordination.
Water molecules are essential for interactions with ATP.

Calmodulin

Calmodulin is a calcium-modulated protein responding to elevated intracellular calcium levels.
It binds to diverse targets (over 300).
Structures of two distinct EF-hand 4 helix bundles joined by a linker.

Calmodulin EF Hands

EF hands are helix-turn-helix motifs.
The turn regions (white) are relatively long and include important amino acids like Asp and Glu, at the end of the second helix.
These residues are essential for calcium binding.
Similar motifs are found in many other proteins.

Calcium Binding

One calcium ion binds to each loop region in helix-loop-helix.
The loop flexibility orients residues towards calcium.
Calcium prefers oxygen ligands (especially carboxylates).
Calcium is coordinated between 2x Asp, Glu, Asn plus a backbone carbonyl oxygen.

Calcium Binding Structural Change

To bind calcium, helices repack, changing arrangement from almost parallel to at right angles.

Ca-Calmodulin Complex Hydrophobic Patch

Rearrangement opens a hydrophobic core within the EF hand.
This exposes a hydrophobic surface.
The favoured energy from calcium binding compensates for exposing hydrophobic residues to water.

Ca-Calmodulin Protein Binding

Ca-Calmodulin binds diverse targets (with exposed nonpolar regions.
EF hands adjust relative position and shape of the cavity.
Methionine positions increase flexibility to optimise packing.
Calmodulin regulates over 300 different proteins, including a calmodulin-dependent kinase.
Forming this complex activates the kinase.

Covalent Modifications

Over 200 covalent modifications are common in eukaryotic proteins, including glycosylation for signal transduction, lipidation for membrane localization, and ubiquitination for degradation.

Protein Glycosylation

Extracellular domains in eukaryotes often have heavily-modified oligosaccharides (up to 20% protein mass).
Glycosylation affects cellular trafficking, and are targets for immune interactions.
Saccharides are added to specific sites (N-linked to Asn; O-linked to Ser/Thr).

Regulatory Modifications

Eukaryotic proteins undergo regulatory modifications by small molecular groups.
This alters activity and interaction with other proteins. –e.g. phosphorylating Tyr, Ser, Thr residues (kinase action); acetylating Lys (on histones).
Modifications may employ whole protein units (e.g. ubiquitin, or SUMO).
Lipid addition can temporarily or permanently target proteins to the membrane.

Function and Structure Constraints

Protein function and structure are related (constraining their sequences).

Protein Mutations

Protein coding sequences are susceptible to mutations.
Mutations from mutagens or DNA replication errors accumulate slowly, even within a species.
Amino acid sequence differences can be present between proteins from different species.
Mutations are often neutral, however, they can impact protein function in the event more apparent differences are found. Evolutionary changes are not randomly distributed.

Active Site Residues

Active site residues are crucial for function and highly conserved.
An enzyme's active site is complementary to the ligand, facilitates catalysis.
Important residues are conserved, and if mutated could reduce efficiency.
Even minor alterations in the specific amino acid sequence can affect substrate specificity.

Buried Residues

Buried residues affect protein stabilization and key element positioning.
Commonly conserved.
Tolerates substitutions of similar amino acids (e.g. Val to Ile).

Surface Residues

Surface residues (not involved in catalysis or binding) are often more variable.
Surface residues are predominantly involved in protein-protein interactions.
Substitutions are generally tolerated and specific to protein solvent interactions.

Different Sequences for Protein Function

Many residues that don't directly affect function can drift over generations resulting in slight differences in amino acid sequence even within species.
For instance, hen and turkey lysozymes differ by just 5 amino acids, but the main functions are similar. This is an example of differing sequence not substantially impacting the final outcome.

Protein Divergence

Proteins can maintain or acquire new functions by slowing accumulating changes over evolutionary time.
Proteins can vary in function despite sequence similarity.
Switching function is more probable when proteins with existing functions are interacting with only slightly differing substrates.

Substrate Specificity in D-LDH Homologs

D-2-hydroxyisocaproate DH similar to D-LDH, but differ in the substrate it works on (resulting in different precursors).
Changes in D-LDH’s methyl-binding pocket allow accommodation of larger substrates rather than pyruvate (by changes in amino acid structure).
Other family members use varying groups to bind different substrates, thereby, resulting in different methyl-binding pockets.

Residues Differentiate Homologs

Residues lining the methyl pocket are significantly different, distinguishing D-LDH orthologs from related enzymes with dissimilar substrates.

β-Carbonic Anhydrases

Carbonic anhydrases use a zinc-activated water ion to hydrolyse CO₂, forming carbonic acids crucial in various biochemical processes.
The catalytic zinc site comprises a conserved Cys-X-X-Cys motif with an Asp/Arg segment.
A hydrophobic pocket helps to bind CO₂.

β-Carbonic Anhydrase Divergence

Bacterial carbonic anhydrases (epsilon class) exhibit high sequence divergence compared to their pea counterparts (yet maintaining similar structure and active sites).
The active site has a conserved structure although only 5 critical active site residues are highly similar across similar types of enzymes.

Human A/B/O Glycosyltransferases

Human blood groups are determined by the presence of A/B/O antigens.
The differences are due to different glycosyltransferases in the saccharide group (GlcNAc, Glc, or no saccharide) attached to a ceramide pentasaccharide.
Different inactive alleles can give rise to different O-antigen blood groups

GTA vs GTB Glycosyltransferases

These enzymes have slightly different, but highly similar, structures.
GTA transfers N-acetyl glucosamine to the antigen, while GTB transfers glucose to the same antigen.
Differences centre on adding the amide to C₂ in the different substrate types.

GTA vs GTB Active Site

GTA and GTB enzymes (A and B glycolsyltransferases) differ in 4 amino acids.
Leu to Met exchange in amino acid sequence directly impacts the space available to bind amide group.
These minor differences result in important biochemical functional differences.

Protein Divergence and Function

Sequence divergence in protein functional areas is associated with new function formation.
Key changes in local areas (like DLDH) define differences in substrate recognition.
Minor substitutions, such as in GTA and GTB, can lead to changes in function.
Proteins can maintain their function despite notable sequence differences (e.g., in differing types of carbonic anhydrases).

Protein Function Variation with Sequence Similarity

Proteins with the potential to evolve new functions easily show structurally or chemically similar partners.
Minor substrate differences in an existing binding site enable a protein to function with slightly different substrates.
Highly different substrates require many changes in the enzyme for a new function to evolve; for this reason, it is less likely to occur.

Bacterial Enzymes and Diversity

Eukaryotic organisms exhibit similar core metabolisms and enzymatic properties.
Bacteria display enormously diverse metabolisms, with a broader range of enzyme functions to adapt to various molecular environments.
Repeated evolutionary mechanisms lead to evolution of new functions in bacterial enzyme families
Function identification in bacterial enzymes can be quite challenging given their vast diversity.

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