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glyoxylate reductase

Metode spectofotometrice de identificare a enzimei:


The Paracoccus sp. 12-A FDH
The activity of FDHs toward formate was assayed at 30 _ i! 100 m" sodium phosphate
#uffer $pH %.0& co!tai!i!' 1.0 m" (AD) a!d various co!ce!tratio!s of sodium formate.
The activity of FDHs toward '*yo+y*ate was assayed at 30 _ i! ,0 m" sodium "-.
#uffer $pH ,.,& co!tai!i!' 0.1 m" (ADH a!d various co!ce!tratio!s of '*yo+y*ate. /!e
u!it was defi!ed as the co!versio! of 1 *mo* of su#strate per 1 mi!. 0i!etic parameters
were ca*cu*ated from p*ots of v12.3 versus
2.3. The deuterium derivative of formic acid was purchased from .i'ma4
A*drich5 a!d used for determi!atio! of the primary isotope effects o! '*yo+y*ate
reductio! of the wi*d-type a!d muta!t FDHs. Protei! co!ce!tratio!s were ca*cu*ated
usi!' a! e+ti!ctio! coefficie!ts at 260 !m of 7%5330"_1 cm_1 for FDHs5 as determi!ed
from the ami!o acid compositio! a!d mo*ecu*ar wei'ht of Paracoccus sp. 12-A FDH .
8ased o! the 'e!ome se9ue!ce of :hi;o#ium et*i5 the !odu*ati!' e!dosym#io!t of the
commo! #ea! p*a!t5 we
predicted a putative 3-phospho'*ycerate dehydro'e!ase to e+hi#it <HP: activity i!stead.
The protei! was overe+pressed a!d purified. The e!;yme is
homodimeric u!der !ative co!ditio!s a!d is i!deed capa#*e of reduci!' #oth '*yo+y*ate
a!d hydro+ypyruvate. /ther su#strates are phe!y*pyruvate a!d
=eto#utyrate. The hi'hest activity was o#served with '*yo+y*ate a!d phe!y*pyruvate5
#oth havi!' appro+imate*y the same =cat10m ratio. This =i!d of
su#strate specificity has !ot #ee! reported previous*y for a <HP:. The optima* pH for
the reductio! of phe!y*pyruvate to phe!y**actate is pH %. These
data *e!d support to the idea of predicti!' e!;ymatic su#strate specificity #ased o!
phy*o'e!etic c*usteri!'.
The assay mi+ture was i!cu#ated for 1, mi!. ,0 >* from this reactio!
mi+ture was a!a*y;ed o! a 8io:ad Ami!e+ HP?-6%H $300 mm@%.6 mm&
co*um! #y a hi'h performa!ce *i9uid chromato'raph $HPA& $.pectra Physics
.P6600& e9uipped with a varia#*e wave*e!'th BC-visi#*e detector $.P67,0&.
The mo#i*e phase was D m" H* $pH 2&. The f*ow rate was 0.D m* mi!E15 a!d
the e*uate was mo!itored at 210 !m.
A! a*ter!ate spectrophotometric assay for '*yo+y*ate was deve*oped #y Farem#s=i a!d
Hod'=i!so! i! 1GD, 5 #ased o! the co*ored product produced #y the reactio! of
'*yo+y*ate a!d resorci!o* i! the prese!ce of a! acid e+tract of Psuedomonas oxalaticus.
The '*yo+y*ate H rescorci!o* adduct is spectrophotometrica**y visi#*e with a ma+ of
7G0!m5 a!d detectio! *imit of thirtee! micro-mo*ar. I! additio! the co*ored product is
a*so visi#*e #y f*uoresce!ce withi! a pH ra!'e of % to G. This procedure whi*e !ot
e+p*icit*y stated i! the refere!ce is depe!de!t upo! the e!;ymatic activity of '*yo+y*ate
dehydro'e!ase which is a meta#o*ic product of the 'rowth of Psuedomonas only u!der
hi'h o+a*ate co!ditio!s. The Farem#s=i method is re*ia!t o! the o+a*ate supported
Psuedomonas e+tract5 a! e+tract which is e+treme*y time a!d preparatio! i!te!sive to
produce i! a!y apprecia#*e 9ua!tities.
Fi'ure 1. Spectrophotometric assays for glyoxylate. /verview of the
spectrophotometric assays deve*oped for the a!a*ysis of '*yo+y*ate. (A) The ma*ate
sy!thase 1 ma*ate dehydro'e!ase assay5 detectio! is #ased upo! the co!comita!t
reductio! of (ADH a!d o+idatio! P". to yie*d a tetra;o*ium dye. (B) The
'*yco*ate o+idase assay for the stoichiometric productio! hydro'e! pero+ide #ased o!
'*yo+y*ate co!sumptio!. (C) The '*yo+y*ate reductase depe!da!t '*yo+y*ate reductio!
upo! (ADPH o+idatio! visi#*e at 370!m.
The e!;yme-coup*ed assay for '*yo+y*ate was i!itiated #y the additio! of ma*ate sy!thase
a!d ma*ate dehydro'e!ase. The assay co!tai!ed a sta!dard so*utio! of 100 m" T-A-H*
pH %.65 1,0>" 1 6.2,>" "T.1P".5 10m" "'*25 700>" acety*-oA5 ,00>" (AD)5
0-,0>" '*yo+y*ate5 DB1mA ma*ate sy!thase5 a!d DB1mA ma*ate dehydro'e!ase i! a fi!a*
vo*ume of 1mA. The a#sor#a!ce at 7G0 !m was measured after 1 hr i!cu#atio! at 3%J
i! the dar=. The sma** amou!t of "T. reduced for the ;ero '*yo+y*ate co!tro* was
su#tracted from that o#tai!ed i! the prese!ce of '*yo+y*ate.
This cytoso*ic e!;yme p*ays a ro*e i! the meta#o*ism of '*yo+y*ate a!d i! the productio!
of '*uco!eo'e!ic precursors from seri!e via hydro+ypyruvate. I! PH2 '*yo+y*ate a!d
hydro+ypyruvate are meta#o*ised to o+a*ate a!d A-'*ycerate respective*y5 i! reactio!s
#e*ieved to #e cata*ysed #y *actate dehydro'e!ase $ADH&. This accumu*atio! of A-
'*ycerate a!d e+cretio! i!to the uri!e has #ee! re'arded as patho'!omo!ic for PH2 ever
si!ce the first descriptio! of the disease i! 1GD6. However5 two cases have rece!t*y #ee!
descri#ed where A-'*ycericaciduria was !ot a promi!e!t feature su''esti!' that the
disease may #e mis - or u!der-dia'!osed. ertai!*y there is evide!ce that 'roups of
hypero+a*uric patie!ts may i!c*ude some PH2 patie!ts misdia'!osed as PH1 o! the #asis
of disease severity.
rude e+tracts were purified #y !ic=e* affi!ity co*um! 5 e*uti!' recom#i!a!t <:HP:
with ,00m" imida;o*e. Purified protei! was dia*ysed over!i'ht i! T3 dia*ysis mem#ra!e
a'ai!st 20m" potassium phosphate #uffer5 pH%.0 a!d 9ua!tified #y a#sor#a!ce at
260!m.
Bibliografie
.hi!oda T.5 Arai 0.5 Ta'uchi H.5 200%. A hi'h*y specific '*yo+y*ate reductase derived
from a formate dehydro'e!ase.
Fauvart ".5 8rae=e! 0.5 Da!ie*s :.5 Cos 0.5 (dayi;eye ".5 (o#e! K.P.5 :o##e! K.5
Ca!der*eyde! K.5 "ichie*s K.5 200%. Ide!tificatio! of a !ove* '*yo+y*ate reductase
supports phy*o'e!y-#ased e!;ymatic su#strate specificity predictio!.

arpe!ter ..-.5 200D. -!;yme *i!=ed spectroscopic assays for <*yo+y*ateH
The use of Peptidy*'*yci!e a*pha-Amidati!' "o!o+y'e!ase for the discovery of (ove*
a*pha-Amidated hormo!es.
D. P. re'ee!5 -.A. Li**iams5 .. Hu*to!5 <. :ums#y5 2003. "o*ecu*ar A!a*ysis of the
<*yo+y*ate :eductase $<:HP:& <e!e a!d Descriptio! of "utatio!s B!der*yi!' Primary
Hypero+a*uria Type 2