Adenylate cyclase, AC for short, is a membraneIntegrin, be able toATPIntocAMP, causing cell signal responseG proteincouplingIn the systemEffector。Adenylate cyclase usually needs magnesium ion, which may be closely related to the enzyme mechanism.The catalysed cAMP then acts as a regulatory signal through specific cAMP binding proteins (transcription factors, enzymes (such as cAMP dependent kinases) or ion transporters).Adenylate cyclase is widely distributed inmammalOfcell membraneMedium.It plays a key regulatory role in almost all cells.It is also the most known enzyme: six different adenylate cyclases have been described, all of which catalyze the same reaction, but belong to unrelated gene families without known sequence or structural homology.The most famous adenylate cyclases are AC-III, which widely exists in eukaryotes and plays an important role in many human tissues.[2]
Chinese name
Adenylate cyclase
Foreign name
adenylate cyclase
Abbreviation
AC
Type
Membrane integrin
Role
Convert ATP into cAMP
Distribution
Cell plasma membrane, nuclear membrane and endoplasmic reticulum
Hormones and correspondingMembrane receptorAfter binding, it activates adenylate cyclase mediated by G protein.hormone receptorImbedded in cell membraneLipid bilayerInside, the part where it combines with hormone faces the outside of the cell.Adenylate cyclase also residesplasma membraneMiddle, located inside the cell.
Excitatory typeG proteinBefore (Gs) is not activated, G α protein andgross domestic productIt is inactive. Once the receptor is combined with hormone, it can induce G α - GDP on G protein andGTPExchange into G α - GTP in the active state.At the same time, G α - GTP is partially separated from G β - γ and moved to the adjacent β - γ adenylate cyclase site to activate adenylate cyclase, which catalyzes ATP to cAMP.However, the life of G α - GTP is short, because G α itself has GTPase activity, which can hydrolyze GTP into GDP and Pi.It becomes inactive G α - GDP again, and returns to combine with G β γ to form a complete inactive G protein (a - β γ) - GDP.
classification
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Class I adenylate cyclase (AC-I)
The first type of adenylate cyclase exists in Escherichia coliE. coliAnd many other bacteria (such as CyaA P00936).This is the first class of adenylate cyclases described.It has been observed that glucose deficient Escherichia coli produces cAMP as an internal signal to activate gene expression for absorption and metabolism of other sugars.CAMP plays this role by binding to the transcription factor CRP (also called CAP).AC-I is a macromolecular cytoplasmic enzyme (about 100kDa) with a large regulatory domain (about 50kDa) for indirect detection of glucose levels.As of 2012, AC-I has not obtained the crystal structure, but some indirect structural information has been obtained.It is known that the half near the N-terminal is the catalytic part, which requires two Mg2+ions.S103, S113, D114, D116 and W118 are five indispensable residues.The AC-I catalytic domain and the palm domain of DNA polymerase β belong to the same superfamily.[1]
Class II adenylate cyclase (AC-II)
These adenylate cyclases are pathogenic bacteria (e.g. Bacillus anthracisBacillus anthracis, Bordetella pertussisBordetellapertussis, Pseudomonas aeruginosaPseudomonas aeruginosaAnd Vibrio vulnificusVibrio vulnificus）Toxins secreted during infection, such as anthrax toxin.These bacteria also secrete proteins that allow AC-II to enter host cells, and then exogenous adenylate cyclase destroys normal cellular physiological processes.The gene of AC-II is called cyaA.Several crystal structures of AC-II enzyme are known.
Class III adenylate cyclase (AC-III)
Due to the important role of the third type of adenylate cyclases in human health, they are based on extensive research and therefore become the most familiar type of adenylate cyclases.They also exist in some bacteria, especially Mycobacterium tuberculosisMycobacterium tuberculosisThey seem to play a key role in the pathogenesis of Mycobacterium tuberculosis.Most AC-III are integral membrane proteins, which are involved in the process of converting extracellular signals into intracellular reactions.Earl Sutherland discovered the key role of AC-III in human liver - adrenaline indirectly stimulates adenylate cyclase to mobilize stored energy in the "fight or flight" response, and therefore won the Nobel Prize in 1971.The role of adrenaline is to transmit chemical signals from the outside of the cell through the membrane to the inside of the cell (cytoplasm) through the G protein signal cascade.The external signal (adrenaline in this case) binds to the receptor, and the receptor transmits the signal to G protein, which transmits the signal to adenylate cyclase, which transmits the signal by converting adenosine triphosphate into AMP.
CAMP is an important molecule in eukaryotic signal transduction, namely the so-called second messenger.Adenylate cyclase is usually activated or inhibited by G protein, which is coupled with membrane receptors, so it can respond to hormones or other stimuli.After the activation of adenylate cyclase, the cAMP produced as the second messenger interacts with other proteins such as protein kinase a and cyclic nucleotide gated ion channels and regulates them.
The light activated adenylate cyclase (PAC) is found in Euglena gracilisEuglena gracilisIt can be expressed in other organisms through gene manipulation.Blue light emitted to cells containing PAC will activate it, thus rapidly improving the conversion rate of ATP to cAMP.This is an important technology for neuroscience research, because it can enable them to rapidly improve the level of cAMP in specific neurons, and study the impact of increased neural activity on biological behavior.By modifying the nucleotide binding site of rhodopsin guanylyl cyclase, researchers recently designed a green light activated rhodopsin adenylate cyclase (CaRhAC).[3]
structure
Most AC-III are transmembrane proteins with 12 transmembrane segments.It consists of six transmembrane segments, followed by the C1 cytoplasmic domain, then another six membrane segments, and finally the second cytoplasmic domain C2.The important part of its function is the N-terminal and C1 and C2 areas.The C1a and C2a sub domains are homologous and form intramolecular "dimers" as active sites.In Mycobacterium tuberculosis and many other bacteria, the AC-III polypeptide is only half long, including a six transmembrane domain, followed by the cytoplasmic domain, but two of them form functional homodimers, similar to the mammalian AC-III structure with two active sites.In non animal AC-III, the catalytic active cytoplasmic domain is linked to other (not necessarily transmembrane) domains.
The third type of adenylate cyclase domain can be further divided into four subfamilies, called IIIa to IIId.Membrane bound ACs of animals belong to Class IIIa.
mechanism
The reaction requires two metal cofactors (Mg or Mn) to coordinate with two aspartic acid residues on C1.They perform a nucleophilic attack on the 3 '- OH group of ribose on the α - phosphate group of ATP.The two lysine and aspartate residues on C2 select ATP instead of GTP as the substrate, so this enzyme is not guanylate cyclase.A pair of arginine and asparagine residues on C2 are responsible for stabilizing the transition state.However, in many proteins, these residues mutate while adenylate cyclase remains active.
Subtype
There are ten known adenylate cyclase subtypes in mammals: ADCY1-ADCY10.(Sometimes there are Roman numerals, which should not be confused with the Roman numerals indicating the type of adenylate cyclase (such as AC-III).)The main difference lies in the regulated mode, and it is differentially expressed in different tissues during mammalian development.
Regulation
Adenylate cyclase is regulated by G protein, which can exist in the form of monomer or heterotrimer and is composed of three subunits.The activity of adenyl cyclase is controlled by heterotrimeric G protein.When the complex is composed of α, β and γ subunits and GDP is combined with α subunit, G protein is inactive or inhibitory.In order to activate G proteins, ligands must bind to receptors and cause conformational changes.This conformational change causes the α subunit to separate from the complex and bind to GTP.This G - α - GTP complex then binds to adenylate cyclase and causes cAMP activation and release.The signal is turned off, that is, the mechanism of adenosine cyclase deactivation and inhibition of cAMP. The inactivation of the active G α - GTP complex caused by GTP hydrolysis is completed quickly. This reaction is catalyzed by the intrinsic enzyme activity of GTPase located in the α subunit.It is also regulated by tricholin and other subtype specific effectors:
·Isoenzymes I, III and VIII were also stimulated by Ca2+/calmodulin.
·Ca2+inhibits V and VI isoenzymes in a calmodulin independent manner.
·Isozymes II, IV and IX are stimulated by the alpha subunit of G protein.
Homologous I, V, and VI are most significantly inhibited by Gi, while other homotypes are less subject to dual regulation of inhibitory G protein.
·Soluble AC (sAC) is not a transmembrane form, and is not regulated by G protein or orodin, but acts as a bicarbonate/pH sensor.It anchors at different locations in the cell and forms a local cAMP signal domain with phosphodiesterase.
In neurons, calcium sensitive adenylate cyclase is located beside the calcium channel to accelerate the response to Ca2+influx;They may play an important role in the learning process. One of the evidences is that adenosine cyclase is coincidence detectors, which means that they are only activated by several different signals occurring together.In peripheral cells and tissues, adenylate cyclase seems to form molecular complexes with specific receptors and other signal proteins in a subtype specific manner.
Class IV adenylate cyclase (AC-IV)
AC-IV for the first time in Aeromonas hydrophilaAeromonas hydrophilaYersinia pestis found inYersiniapestisAC-IV has also been reported.They are the smallest adenylate cyclases;AC-IV (CyaB) from Yersinia is a dimer composed of 19 kDa subunit, and there is no known regulatory component.AC-IV forms a superfamily with the Mary thiamine triphosphatase called CYTH (CyaB, thiamine triphosphatase).
Class V and VI adenylate cyclase (AC-VIV)
These two forms of adenylate cyclase are only found in certain bacteria (such as Prevotella ruminalisPrevotella ruminicolaHecai bean rhizobiaRhizobium etli）It has been reported in and has not been widely described.Class VI enzymes have catalytic centers similar to those of Class III.
Experimental methods
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Electron microscopic enzyme cytochemical test method for adenylate cyclase
Howell et al. (1972) first usedImineAdenosine diphosphate(AMP-PNP) as substrate, successfullyAnimal cellAdenylate cyclase was located in.It has been confirmed that ATP PNP is the adenylate cyclaseSpecificitySubstrate, under the action of adenylate cyclase, can generate cAMP and PNP, the latter is related to lead captureIonic reaction, forming an insolubleelectron densityVery high product.But lead can restrainenzymatic activityIn order to overcome this shortcoming, Fujiomto et al. (1981) designed to use potassiumSulfoxide（DMSO）OfLead nitrateLaw.The following is the lead nitrate method of DMSO used by Fujimoto et al.
Stationary liquid formula: More than 2%PolyoxymethyleneAnd 0.25%glutaraldehydeMixed liquid。For liver, more than 1% glutaraldehyde fixation can lead to obviousEnzyme inactivation。However, if 5% DMSO was added, the enzyme activity could be well preserved.In general, the concentrations of paraformaldehyde and glutaraldehyde should vary from tissue to tissue.Generally, it is fixed at 0~4 ℃ for 2 hours or at room temperature for 1 hour.
wash: Rinse for 1 hour~overnight with the buffer solution with the same fixed solution, and replace the buffer solution several times during the period;Then thick section was made;
Incubation: See ref for formula and conditions{table:Enzyme-7},
When AMP-PNP, as the specific substrate of adenylate cyclase, is stored at room temperature or for a long time, compounds such as ATP, AMP-PN and AMP-PNP-P tend to increase, which should be noted.The addition of levamisole is due to the fact that the substrate AMP-PNP is susceptible to tissuealkaline phosphataseDecomposition. The addition of levamisole can inhibit the activity of this enzyme. In tissues with high alkaline phosphatase activity,LevamisoleFactors such as concentration of.Sodium fluorideAdenylate cyclaseactivator, which should be carried out according to different organizationsPretreatmentThen, add sodium fluoride into the incubation solution.
The remaining DMSO can be connected withOsmic acidReactionDiffusivityFine particles.After fixation, 1%~2% osmic acid was prepared with sodium dimethyl arsenate buffer solution.After osmic acid fixationEnzyme reactionThe product has a certain impact. Some people think that the fixed time should not exceed 10min, which may cause the enzyme reaction product particles to become coarser, larger and diffuse. Sometimes there is no reaction product due to complete dissolutionNegative results。Therefore, the fixation time with osmic acid should be controlled within 5 minutes.2~4% glutaraldehyde is also used to replace osmic acid for post fixation.
