Thesis (MSc) Stellenbosch University, 2014 / ENGLISH ABSTRACT: In this study, the effect of the amino acid residues at positions 40 and 407 on the catalytic activity of
porcine CYP17A1 was investigated. Porcine cofactor CYB5 was cloned from porcine liver tissue and
its effect on the catalytic activity of porcine CYP17A1 was determined. The influence of rat, human
and angora CYB5 on the lyase activity of porcine CYP17A1 was subsequently determined and
compared to the influence of porcine CYB5. Wt porcine CYP17A1, which has residues Val40 and
His407, catalysed the conversion of prog efficiently with ~50% prog converted to 17OHprog (~40%)
and A4 (~10%) after 3 hr. After 24 hr, negligible levels prog remained with ~71% 17OHprog and
~25% A4 being produced. Low levels of 16OHprog were formed (~9%). The Leu105Ala mutation
reduced wt 17α-hydroxylase activity, with 70% prog remaining after 24 hr while 16OHprog (~10%)
levels remained unchanged. Porcine CYP17A1 with residues Leu40 and His407, exhibited similar
catalytic activity towards prog as did wt porcine CYP17A1 (Val40 and His407 residues), while porcine
CYP17A1 with residues Leu40 and Leu407 increased the formation of A4 2-fold to 54% at 24 hr and
porcine CYP17A1 with residues Val40 and Leu407 resulted in the highest formation of A4 (90%). Wt
porcine CYP17A1, while having converted 95% of the prog substrate, produces only ~16% A4 after 24
hr. In the presence of porcine CYB5, however, the lyase activity was stimulated with 85% of prog
being converted to A4 and only 13% 17OHprog remaining. The lyase activity was also stimulated by
CYB5 from other species, resulting in an increase in A4 production of 60.6%, 24% and 11.6% by rat,
angora and human CYB5, respectively. The degree of lyase stimulation correlated to the percentage
identity of the CYB5 amino acid sequences to porcine CYB5. While the Val and Leu residues at
position 40 do not appear to influence the lyase activity of porcine CYP17A1 as prominently as the
residue at position 407, it is the charged residue at 407 that plays a significant role in the production of
A4, decreasing A4 production irrespective of the Val and the Leu residues at position 40. It would,
furthermore, appear that the stimulation of lyase activity of CYP17A1 is the greatest when assaying
this activity in the presence of CYB5 of the same species as was detected when co-expressing porcine
CYP17A1 and porcine CYB5. / AFRIKAANSE OPSOMMING: In hierdie studie is die invloed van die aminosuurresidue by posisies 40 en 407 op die katalitiese
aktiwiteit van vark CYP17A1 ondersoek. Vark CYB5 is geklooneer vanuit vark lewer weefsel en die
effek van hierdie kofaktor op die katalitiese aktiwiteit van vark CYP17A1 is bepaal. Die invloed van
rot, mens en angora CYB5 op die liase aktiwiteit van vark CYP17A1 is daarna bepaal en vergelyk met
die invloed van vark CYB5. Vark CYP17A1-VH, (kodeer Val40 en His407), kataliseer die
omskakeling van prog doeltreffend met ~50 % prog wat omgeskakel word na 17OHprog (~40%) en A4
(~10%) na 3 uur. Na 24 uur, is feitlik alle prog omgeskakel, met ~71% 17OHprog en ~25% A4
geproduseer. Lae vlakke 16OHprog is ook gevorm (~9%). Die Leu105Ala mutasie verminder 17α-
hidroksilase aktiwiteit, met 70% prog wat na 24 uur nie omgesit is nie, terwyl 16OHprog (~10%)
vlakke onveranderd gebly het. Vark CYP17A1-LH (kodeer Leu40 en His407), en CYP17A1-VH het
diselfde katalitiese aktiwiteit teenoor prog getoon, terwyl vark CYP17A1-LL (kodeer Leu40 en
Leu407) die vorming van A4 2-voudig verhoog het tot 54% na 24 uur. Vark CYP17A1-VL (kodeer
Val40 en Leu407) se katalitiese aktiwiteit het gelei tot die hoogste vorming van A4 (90%). Alhoewel
CYP17A1-VH, 95% van die prog substraat omgeskakel het is slegs ~16% A4 geproduseer na 24 uur.
In die teenwoordigheid van vark CYB5 is die liase aktiwiteit egter gestimuleer, en is 85% van die prog
substraat omgeskakel na A4 met slegs 13% 17OHprog teenwoordig na 24 uur. Die liase aktiwiteit is
ook gestimuleer deur CYB5 van ander spesies, wat lei tot 'n toename in A4 produksie van 60,6% , 24%
en 11,6% deur rot, angora en menslike CYB5, onderskeidelik. Daar is gevind dat daar’n sterk
korrelasie is tussen die stimulering van die liase aktiwitieit en die persentasie aminosuur volgorde
identiteit van CYB5 afkomstig vanaf die verskillende spesies. Terwyl die Val en die Leu
aminosuurresidu op posisie 40 wel die liase aktiwitiet tot ‘n mate beȉnvloed, blyk dit uit die data dat die
potitief gelaaide residue by 407 'n belangrike rol speel in die produksie van A4, en A4 produksie
verlaag ongeag van die Val en die Leu residu by posisie 40. Dit wil ook verdermeer voorkom asof die
stimulering van die liase aktiwiteit van CYP17A1 die hoogste is wanneer die ensiem gekataliseerde
reaksie deurgevoer word in die teenwoordigheid van CYB5 en CYP17A1 afkomstig vanaf dieselfde
spesies.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/86634 |
| Date | 04 1900 |
| Creators | Fox, Cheryl-Leigh |
| Contributors | Swart, A. C., Storbeck, K-H, Swart, P., Stellenbosch University. Faculty of Science. Dept. of Biochemistry |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xi, 126 p. : ill. |
| Rights | Stellenbosch University |
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