Enolase: Diferenzas entre revisións

UsingUsando isotopicsondas probesisotópicas, theo overallmecanismo mechanismglobal forproposto convertingpara converter o 2-PG toen PEP isé proposed to be anunha [[E1cb|E1cbreacción eliminationde reactioneliminación E1cb]] involvingno aque carbanionestá intermediateimplicado un intermediato [[carbanión]].<ref>{{cite journal |author=Dinovo EC, Boyer PD |title=Isotopic probes of the enolase reaction mechanism |journal=J Biol Chem |volume=240 |issue= |pages=4586–93 |year=1971}}</ref> O Theseguinte followingmecanismo detaileddetallado mechanismestá isbaseado baseden onestudos studiesda ofestrutura crystalcristalina structuree and [[Chemical kinetics|kinetics]]cinética.<ref name=Pancholi/><ref>{{cite journal |author=Poyner RR, Laughlin LT, Sowa GA, Reed GH |title=Toward identification of acid/base catalysts in the active site of enolase: comparison of the properties of K345A, E168Q, and E211Q variants |journal=Biochemistry |volume=35 |issue=5 |pages=1692–9 |year=1996 |month= February|pmid=8634301 |doi=10.1021/bi952186y }}</ref><ref>{{cite journal |author=Reed GH, Poyner RR, Larsen TM, Wedekind JE, Rayment I |title=Structural and mechanistic studies of enolase |journal=Current Opinion in Structural Biology |volume=6 |issue=6 |pages=736–43 |year=1996 |month= December|pmid=8994873 |url=http://linkinghub.elsevier.com/retrieve/pii/S0959-440X(96)80002-9 |doi=10.1016/S0959-440X(96)80002-9}}</ref><ref>{{cite journal |author=Wedekind JE, Reed GH, Rayment I |title=Octahedral coordination at the high-affinity metal site in enolase: crystallographic analysis of the MgII—enzyme complex from yeast at 1.9 Å resolution |journal=Biochemistry |volume=34 |issue=13 |pages=4325–30 |year=1995 |month= April|pmid=7703246 |doi=10.1021/bi00013a022 }}</ref><ref>{{cite journal |author=Wedekind JE, Poyner RR, Reed GH, Rayment I |title=Chelation of serine 39 to Mg2+ latches a gate at the active site of enolase: structure of the bis(Mg2+) complex of yeast enolase and the intermediate analog phosphonoacetohydroxamate at 2.1-Å resolution |journal=Biochemistry |volume=33 |issue=31 |pages=9333–42 |year=1994 |month= August|pmid=8049235 |doi=10.1021/bi00197a038 }}</ref><ref>{{cite journal |author=Larsen TM, Wedekind JE, Rayment I, Reed GH |title=A carboxylate oxygen of the substrate bridges the magnesium ions at the active site of enolase: structure of the yeast enzyme complexed with the equilibrium mixture of 2-phosphoglycerate and phosphoenolpyruvate at 1.8 Å resolution |journal=Biochemistry |volume=35 |issue=14 |pages=4349–58 |year=1996 |month= April|pmid=8605183 |doi=10.1021/bi952859c }}</ref><ref>{{cite journal |author=Duquerroy S, Camus C, Janin J |title=X-ray structure and catalytic mechanism of lobster enolase |journal=Biochemistry |volume=34 |issue=39 |pages=12513–23 |year=1995 |month= October|pmid=7547999 |doi=10.1021/bi00039a005 }}</ref> Cando Wheno the substrate,substrato 2-phosphoglycerate,fosfoglicerato bindsse toune á α-enolase, itso carboxylseu groupgrupo coordinates[[carboxilo]] withcoordínase twocon magnesiumdous ioncofactores cofactorsións inmagnesio theno activesitio siteactivo. Isto Thisestabiliza stabilizesa thecarga negativenegativa chargeno onoxíxeno thedesprotonado deprotonatedá oxygenvez whileque increasingincrementa thea acidityacidez of thedo alphahidróxeno hydrogenalfa. Enolase’sA Lys³⁴⁵ deprotonatesda theenolase alphadesprotona hydrogen,o andhidróxeno thealfa, resultinge negativea chargecarga isnegativa stabilizedresultante byé resonanceestabilizada topor theresonancia carboxylateco oxygenoxíxeno anddo bycarboxilato thee magnesiumpolos ioncofactores cofactorsMg²⁺. Despois Followingda thecreación creationdo ofintermediato the carbanion intermediatecarbanión, theo hydroxideOH onde C3 isé eliminatedeliminado ascomo waterauga withcoa theaxuda help ofdo Glu²¹¹, ande PEPfórmase iso formedPEP.

AdditionallyAdicionalmente, conformationalno changesencima occurteñen withinlugar thecambios enzymeconformacionais thatquw aidaxudan catalysis.á catálise. In humanNa α-enolase humana, theo substratesubstrato isestá rotatedrotado intona positionsúa uponposición bindingao tounirse theao enzymeencima duedebido toás interactionsinteraccións withcos thedous twoións catalyticmagnesio magnesium ionscatalíticos, coa [[GlutamineGlutamina|Gln]]¹⁶⁷, ande Lys³⁹⁶. Os Movementsmovementos ofdos loopsbucles [[SerineSerina|Ser]]³⁶ toa His⁴³, Ser¹⁵⁸ toa [[GlycineGlicina|Gly]]¹⁶², andw [[AspartateAspartato|Asp]]²⁵⁵ toa [[AsparagineAsparaxina|Asn]]²⁵⁶ allowpermiten que a Ser³⁹ tose coordinatecoordine withco Mg²⁺ ande closebloquee offo thesitio active siteactivo. Ademais In addition to coordinationde withcoordinarse thecos catalyticións magnesiummagnesio ionscatalíticos, theo pKa ofdo thehidróxeno substrate’salfa alphado hydrogensubstrato isrebáixese alsotamén lowereddebido dueá toprotonación protonationdo ofgrupo thefosforilo phosphorylpola groupacción byda His¹⁵⁹ ande itsa proximitysúa toproximidade á Arg³⁷⁴. A Arg³⁷⁴ alsotamén causescausa que a Lys³⁴⁵ indo thesitio activeactivo sitesexa todesprotonada, becomeo deprotonated,cal which''ceba'' primesou prepara á lisina Lys³⁴⁵ forpara que exerza itso roleseu inpapel theno mechanismmecanismo.