Module 5
Feedback Inhibition
(end product inhibition) When enough product accumulates to a point above which binding to an enzyme and it inhibits its activity
Describe Dr. Edelmann's Salmonella PhoP/PhoQ example of phosphorelays
2 component regulatory system to serve as a transcriptional regulator responding to magnesium starvation in S. typhimurium Allows Salmonella to sense the transition from extracellular environment to inner cellular location in the host cell Virulence factors that allow survival in intracellular space which are activated by the PhoP/PhoQ system
Proteasome as a protein degradation machine in eukaryotes
9S regulatory particle (RP) binds to either both ends of the 20S core particle (CP) to form the 26S proteosome. 1 9S RP contains AAA+ ATPases and has several functions, including: Removal of ubiquitin modification from substrates Recognition of proteins that are targeted for degradation Protein unfolding 20S CP-active sites are responsible for protein degradation, which is a chamber containing active sites
How does the value of Km effect enzymatic reactions?
A small Km means a high affinity of an enzyme since it means a reaction can reach half of the max velocity in a small number of substrate concentration Enzymes with small Km will reach Vmax faster than one with a larger Km In regulatory enzymes, positive cooperativity creates a sigmoidal equation
Describe Dr. Edelmann's example for Allosteric Control Mechanisms
ADP and AMP regulate the reaction between fructose 6-phosphate (F6P) and fructose 1,6-bisphosphate (FBP). Phosphofructokinase (PFK) is responsible for adding a phosphate to a F6P to create FBP. Fructose 1,6-phosphatase is responsible for dephosphorylating FBP to F6P. If PFK is active, glycolysis will progress. If F16P is active, then we proceed towards gluconeogenesis. AMP is an allosteric inhibitor of the dephosphorylation of FBP to F6P. AMP binds to F16P and allosterically inhibits it so that it cannot remove the phosphate from the FBP. In the presence of AMP, there's an increase in FBP which leads to increased glycolysis under conditions of low energy charge.
FtsH (Zn2+ - metalloproteases)
ATP-dependent metalloprotease Function in quality control of integral membrane proteins and LPS biosynthesis
Lon protease
ATP-dependent protease (AAA+ protease) Important in proteolysis Can regulate gene expression
Lysine Acetylation in Prokaryotes
Acetylation is involved in central metabolic pathways Enzymes that regulate the following processes have their histones modified: glycolysis, gluconeogenesis, the TCA cycle, glyoxylate bypass, glycogen biosynthesis, amino acid biosynthesis, fatty acid metabolism, etc...
Phosphorylation as a form of Post-Translational Modification
Attachment of a phosphate onto the functional groups of an amino acid side chain Hydroxyl group is modified by phosphorylation from an ATP, creating an ADP Phosphorylation can activate or inactivate an enzyme depending on the site of phosphorylation
Recognition-Motif Dependent Proteolysis
Attachment of protein modifiers is not the only way for proteins to be targeted for proteolysis Can also be caused by a recognition motif in the protein sequence Primary sequence of a protein has an impact on the proteolytic cleavage of a protein mportant in pathogenic bacteria, where they can recognize the stress of their environment and react accordingly within a host. It could decide to secrete exopolysaccharides to protect the bacteria and contribute to pathogenicity
Sigma Factors and their role in Metabolic Regulation on a transcription level
Bacteria (NOT archaea!)- have different sigma factors, which associate with RNA polymerase core to mediate promoter recognition Proteins which bind directly with RNA polymerase and help it to bind to very specific promoter sequences Sigma factors are present in all bacteria E. Coli has several sigma fators, which enable specific binding of RNA polymerase to gene promoters. Because of this, it can fine tune its transcriptional output
Describe cAMP-CPR Complex and catabolic Repression
CAP= catabolite activator protein CRP= catabolite regulation protein The presence or absence of glucose determines the concentration of cAMP, the small molecule effector that determines the activity of CRP As PTS sugars (glucose) are exhausted, cAMP levels rise and bind to CRP CRP dimer is activated by binding 2 molecules of cAMP, causing a conformational change, allowing CRP to bind to specific pseudo-palindromic DNA in gene operators and mediate regulation
Describe how Two-component System and Sigma Factors can Work together
Can be switched on and off to facilitate different gene expression, therefore increasing survival of an organism Cell envelope undergoes stress. Sigma factors and histokinases are either inhibited or regulated, resulting in different gene expressions
Ubiquitin (Ub) Modification in Eukaryota
Characterized by the addition of ubiquitin molecules to a lysine residue of protein substrate Common modification in eukaryotes (Not in prokaryotes, ubiquitin is not present) Can mark proteins for degradation Can play non-proteolytic functions Regulation of endocytosis, DNA repair, intracellular trafficking, signal transduction, etc.
The 2 forms of metabolic regulation
Constitutive expression, Protein amount or activity needs to be regulated for some proteins
Bacteria have 2 unrelated modes to regulate enzymatic reactions
Control of regulation of enzymatic activity (feedback inhibition and end product inhibition) Control of regulation of enzyme synthesis (End product repression which functions in the biosynthetic pathways and Catabolic repression which can regulate degradation pathways)
Carbon catabolite repression Mediated by Cra- Independent of cAMP
Cra: multifunctional transcriptional regulator common to enteric bacteria Effects virulence in Shigella and Salmonella Present in gram negative bacteria It is a transcription factor which uses variances in sugar concentrations to activate or inhibit expression of target genes High glucose: Cra is inactivated by PTS uptake of catabolites (ex: glucose) Low glucose: Cra activates transcription of genes for gluconeogenesis, and represses transcription of genes for glycolysis
How does arabinose affect transcription?
E. Coli can use arabinose as a carbon source by converting into xylulose 5-phosphate (an intermediate in pentose pathway), utilizing ribulose 5-phosphate isomerase (encoded by Ara A, AraB, and AraD). AraC can serve as a negative or a positive regulator of this system. It regulates its own synthesis and represses transcription of AraC gene when its concentration exceeds a certain amount. Also positively and negatively regulates AraA,B,D. When there is no arabinose in the environment, AraC binds to the operator and inhibits transcription (it looks like a crochet chain folded in on itself). When arabinose is present, the sequence "relaxes" and undergoes a conformational change and activates transcription Acting as a homodimer, the product of AraC can either bend or let DNA relax which changes the ability of DNA polymerase to access the operator region
What are the enzymes associated with ubiquitin modification in eukaryotes, and what are their functions?
E1- ubiquitin activating enzyme E2- ubiquitin conjugating enzyme E3- ubiquitin ligase DUB- deubiquitinase (can reverse the ubiquitin modification reaction)
Michaelis-Menten equation
Enzyme kinetics v= velocity of reaction Km= substrate concentration at half of the max velocity (½ Vmax)
The Lac Operon: Carbon Catabolite Repression by cAMP-CRP
High levels of glucose result in low concentrations of cAMP , which binds to CRP causing transcriptional activation and modulation of gene expression in the lac operon, which leads to expression of LacI. LacI binds to promotior and represses transcription by RNA polymerase, and this can be relieved upon inducer binding, such as IPTG binding to LacI , which dissociates from the promotor. A LacI can be inactivated by an inducer, but RNA polymerase still requires cAMP/CRP to bind and stimulate transcription for the metabolism of lactose
Methylation as a form of Post-Translational Modification
In eukaryotic organisms, histones are the targets of methylation Addition of methyl groups to amino acids N-methylation of Lysine, Arginine, and Glutamine, O-methylation of the side chain carboxyl of glutamic acid
Describe an example of phosphorylation as a form of Post-translational modification
Isocitrate dehydrogenase (IDH) becomes phosphorylated when there is high concentration of acetate, which leads to its inactivation and reduction of carbon flow (see the TCA cycle)
Allosteric Control Mechanism
Key metabolic enzymes can be controlled allosterically - they change their conformation upon effector binding, which causes change in the apparent binding affinity at a substrate binding site "On" and "off" position which can be switched based on the conformation of a protein There are allosteric activators and inhibitors
Two-Component Regulatory Systems and Phosphorelays
Most histokinases are membrane-bound proteins which have extracellular input domain while response regulators are cytosolic Phosphorelay uses a regulator of an intermediate response, which does not have output domain
Acetylation as a form of Post-Translational Modification
N-Acetylation- addition of an acetyl group to the ε-amines of Lysine side-chains (Nε-acetylation) or the 𝛼-amines of protein N-termini (N𝛼-acetylation)\ There is a charge reduction, but there is no protonation Lysine acetylation is regulatory and reversible (N-acetyltransferases attach it and deacetylases remove it) and occurs in prokaryotes N𝛼-acetylation is not regulatory It is rare in bacteria, but common in Eukarya and Archaea
What are the three ubiquitin-like protein modifiers in prokaryotes?
PUP, TtuB, and SAMP
Describe Dr. Edelmann's Yersinia example of Phosphorelays
Pathogenic Yersinia (bubonic plague and other more relevant diseases) are good for understanding and studying bacteria-host cell interplay Yersinia uses sigma factors, two-component regulatory system, and Rcs phosphorelay systems to respond to extracytoplasmic stress CpxA and RcsC are used to initiate transduction of phosphate through cytoplasmic core response regulation
Two Component Regulatory System
Prokaryotic signal transduction is often regulated by a two component system which functions as a result of phosphate transport between two key proteins Sensor/ sensor-histokinase protein Response regulator protein
How is methylation involved in flagellar movement?
Receptor complexes in clockwise signaling activate CheA producing high levels of phospho-CheY Receptors in the counterclockwise signaling deactivate CheA, resulting in low levels of phosphorylated CheY. This behavior of flagellum motor reflects the eratic proportion of signaling complexes of the kinase-on and kinase-off conformation. In addition, methylation can also be involved, shifting receptor signaling states from one state to another, more methyl groups shift the receptors to "on," restoring balance to the two states and restoring tumbling
Regulation by post-translational modifications (PTM)
Regulates enzymatic activity, and stability Examples: phosphorylation, methylation, adenylation, ADP-ribosylation, Ubiquitin-like modifications
Proteolysis
Regulates enzyme stability Might depend on recognition motif and/or energy
Briefly describe Regulation by Post Translational Modification
Reversible covalent attachment of chemical groups (such as methylation, phosphorylation, acetylation, adenylation, and uridylation) or small protein modifiers (such as ubiquitin-like proteins) can result in PTM PTM usually requires energy, such as in the form of ATP (phosphorylation, adenylation, and acetylation), NAD+ (ADP-ribosylation), and UTP (uridylation) PTM are widespread
Proteases in Prokaryota
Similar to proteasomes in that they're self-compartmentalized All have chamber-like structures where the proteolytic active site are present so only a specific protein can be degraded in these proteases An enzyme gets targeted to the complex in some way, which follows unfolding and eventually degradation of the protein It's still unclear how, but research suggests attachment of small proteins modifiers such as ubiquitin-like modification
Allosteric regulation
Stimulates or inhibits regulatory enzymes Regulates enzymatic activity and conformational changes Can include interaction with another protein or molecule pH
Transcriptional Control of Anabolic Processes- TrpR
The repressor for the Trp operon is produced upstream by TrpR gene, which is constitutively expressed at a low level. TRP monomers become Trp dimers, so these repressors bind to tryptophan and cause a change in repressor conformation allowing the repressor to bind to the operator. This binding of TrpR to operator prevents RNA polymerase from binding to it, so the operon won't be transcribed, and tryptophan isn't produced by its precursor When tryptophan is not present, the repressor is in its inactive conformation, and it can't bind to the operator region so transcription is not inhibited by the repressor
Carbon Catabolite Repression mediated by CcpA
This repression allows cells to choose among several available carbon sources CcpA is a phosphotransferase system (PTS) histidine protein common to low GC Gram-positive bacteria CcpA:HPrSer~P complex: Binds DNA operator Cre elements Can act as both an activator and a repressor of gene expression Ensures hierarchical and efficient use of preferred sugars
Briefly discuss ubiquitin chains
Ubiquitin can form 8-subunit chains: K6, K11, K27, K29, K33, K48, and K63 C-terminus of distal moiety of ubiquitin is attached to one of the 7 lysine residues in ubiquitin Different residues can have different effects on protein substrates K48 are involved in proteolysis by attaching to a substrate and is recognized by a proteasome and is subject to proteolytic degradation (26S proteasome) Bacteria and archaea do not have ubiquitin, but some pathogenic bacteria code for enzymes which control eukaryotic ubiquitin modification such as E3 and DUB
How is the concentration of cAMP determined by the availability of glucose?
When glucose is present, EIIA protein inhibits non-PTS sugar transportation PTS refers to the phosphotransferase system, which is a translocational system for sugar Because of this inhibition, adenylate-cyclase has low enzymatic activity, which lowers cAMP levels When glucose is absent, phosphorylated EIIA enzyme stimulates adenylate-cyclase to generate more cAMP Because CRP dimer binds to cAMP, this changes conformation of DNA, allowing specific gene sequence binding to regulate transcription
Describe an example of lysine acetylation in prokaryotes
_acetyl coA synthetase (ACS)PAT enzyme acetylates ACS, and CobB deacetylates inactive ACS using NAD+Acetate can be converted to an intermediate ecetyl-AMP using ACS and eventually into acetyl CoACobB
Describe RseA as a form of recognition motif dependent proteolysis
anti-sigma factor protein can bind sigma factors in a resting state (which can't bind to RNA polymerase) but when there is stimuli, anti-sigma factor activity becomes relieved
Metabolic regulation is often on...
metabolic branch points It is often irreversible, so a secondary control is often needed to ensure reaction efficiency
Describe SAMP
not homologous to ubiquitin in terms of amino acid sequence, but similar to TtuB. They can be similar to ubiquitin in compact globular beta-grasp folds (aka structural similarities); covalent bonds are linked to lysine-residue targets Sulfur carriers
Proteasome as a protein degradation machine in prokaryotes
only 20S CP is present. Proteasomes are found in all archaea, and in bacteria they are found in the orders of Actinomycetales and Nitrospirales They have associated AAA+ ATPases Example: archaea have proteasome-activating nucleotidase (Pan), AMA, and Cdc484
Describe PUP
prokaryotic and ubiquitin-like protein seen in actinobacteria; intrinsically disordered protein which is covalently linked to proteins via pupylation Different from ubiquitin in structure, sequence, and intrinsically disordered PUP tags proteins for degradation by proteosomes, including metabolism related proteins
Describe DegS as a form of recognition motif dependent proteolysis
protease cleaves anti-sigma factor at one site in response to environmental stress. Cleavage depends on primary sequence of RseA protein. A second protein RseP comes in and cuts the sigma-factor at a second site, thereby relieving RsaA of ant-sigma factor activity
Constitutive Expression
proteins/ genes needed at the same level under all growth conditions Controlled/ efficient processes Indispensable Rely on post-translational controls
Describe TtuB
ubiquitin-like protein which functions as a sulfur carrier in tRNA t-uridine synthesis, and it is covalently attached to target proteins most likely by the C-terminus in glycine
Positive Regulation
when a precursor stimulates an enzyme downstream the pathway
One Component Regulatory System
widely distributed and ancient system in which genetic transcription of operon sequences uses one-component Signal is initiated, the the regulator sends out a response Direct infusion of an input domain to an output domain in a single protein molecule
