Mechanismen enzymatischer Redoxreaktionen
Derivate des Vitamins B2 oder Riboflavins sind als sogenannte Kofaktoren in einer Vielfalt von Enzymen enthalten. Diese (Flavo)proteine sind in der Natur ubiquitär anzutreffen und üben speziell bei metabolischen Prozessen, die Redox Umwandlungen beinhalten, Schlüsselrollen aus. Die Spaltung von (kinetisch) stabilen C-H Bindungen (Dehydrogenierung) und die Übertragung der in diesen Bindungen enthaltenen Energie auf geeignete Akzeptoren sind grundlegende Schritte deren Chemismus noch nicht im Detail verstanden wird. Hierfür sind im letzten Jahrzehnt mehrere mechanistische Vorschläge gemacht worden, die sich aber bisher noch nicht haben bestätigen lassen. Wir untersuchen diese Fragen und verwenden hierzu Enzyme aus der Klasse der D-Aminos¿ure Oxidasen und a-OH-Säure Oxidasen. Wir haben hierbei bereits einige grundlegend neue Erkenntnisse gewonnen. So scheint im
geschwindigkeitsbestimmenden Schritt der Dehydrogenierung ein Hydrid von der Substrat alpha-Stellung auf Position N(5) des oxidierten Flavins übertragen zu werden. Diese Thematik wird in Zusammenarbeit mit der Gruppe der Proff. Pilone / Pollegioni (Varese, Italien) bearbeitet.
Vitamin b2 or riboflavin in its modified forms FMN and FAD is the cofactor of the flavoprotein enzyme family. These enzymes are ubiquitous and, in general, catalyze redox processes. The cleavage of (kinetically) stable C-H bonds and the transfer of the resulting redox equivalents to appropriate acceptors (dehydrogenation) is a key step in flavoprotein catalysis. In spite of its basic importance, its chemical mechanism has been elucidated only in specific cases (e.g. NAD(P)H dehydrogenases). We
study the mechanism of these conversions using enzymes from the family of the D-amino acid oxidases and a-OH acid oxidases/dehydrogenases and have gained some new insights recently. Thus the alpha-H of the substrates of these enzymes appears to be transferred to the position N(5) of the flavin cofactor as a hydride. This project is being pursued in collaboration with the groups of professors Pilone / Pollegioni
- FB Biologie
|(1997): On the mechanism of D-amino acid oxidase : structure/linear free energy correlations and deuterium kinetic isotope effects using substituted phenylglycines Journal of Biological Chemistry. 1997, 272(8), pp. 4924-4934. ISSN 0021-9258. eISSN 1083-351X. Available under: doi: 10.1074/jbc.272.8.4924||
On the mechanism of D-amino acid oxidase : structure/linear free energy correlations and deuterium kinetic isotope effects using substituted phenylglycines
The kinetic mechanism of the reaction of D-amino acid oxidase (EC 220.127.116.11) from Trigonopsis variabilis with [α-1H]- and [α-2H]phenylglycine has been determined. The pH dependence of Vmax is compatible with pKa values of ≈8.1 and >9.5, the former of which is attributed to a base which should be deprotonated for efficient catalysis. The deuterium isotope effect on turnover is ≈3.9, and the solvent isotope effect ≈1.6. The reductive half-reaction is biphasic, the first, fast phase, k2, corresponding to substrate dehydrogenation/enzyme flavin reduction and the second to conversion/release of product. Enzyme flavin reduction consists in an approach to equilibrium involving a finite rate for k-2, the reversal of k2. k2 is 28.8 and 4.6 s-1 for [α-1H]- and [α-2H]phenylglycine, respectively, yielding a primary deuterium isotope effect ≈6. The solvent deuterium isotope effect on the apparent rate of reduction for [α-1H]- and [α-2H]phenylglycine is ≈2.8 and ≈5. The rates for k-2 are 4.2 and 0.9 s-1 for [α-1H]- and [α-2H]phenylglycine, respectively, and the corresponding isotope effect is approx 4.7. The isotope effect on α-H and the solvent one thus behave multiplicatively consistent with a highly concerted process and a symmetric transition state.
|(1993): Kinetic mechanism of D-amino acid oxidases from Rhodotorula gracilis and Trigonopsis variabilis Journal of Biological Chemistry. 1993, 268(19), pp. 13850-13857||
The reaction of two D-amino acid oxidases from the yeasts Rhodotorula gracilis and Trigonopsis variabilis with the substrates alanine and valine in their 2-1H and 2-2H forms was studied employing the stopped- flow spectrophotometric technique. The turnover numbers at infinite substrate and oxygen concentrations were: 20,700/4,250 and 1,730/360 ([2-1H]/[2-2H]alanine and valine, respectively) for the Rhodotorula and 3,150/440 and 2,500/520 ([2-1H]/[2-2H]alanine and valine, respectively) for the Trigonopsis enzymes. The rates of anaerobic enzyme flavin reduction were 20,100/4,000 and 1,820/350 ([2-1H]/[2-2H]alanine and valine, respectively) for the Rhodotorula and 3,470/350 and 2,460/480 ([2-1H]/[2-2H]alanine and valine, respectively) for the Trigonopsis enzymes. The isotope effects on enzyme reduction were 5.0 and 5.2 for Rhodotorula and 9.9 and 5.1 for Trigonopsis D-amino acid oxidases with alanine and valine, respectively. This suggests that the intrinsic isotope effect on rupture of the substrate α-C-H bond can be as high as 10. The rate-determining step corresponds to the enzyme reductive half-reaction in contrast to the mammalian kidney enzyme where it is the product release from oxidized enzyme (Massey, V., and Gibson, Q.H. (1964) Fed. Proc. 23, 18-29). Upon anaerobic reaction with substrate, the yeast enzymes do not form the transient long wavelength absorbing species which are characteristic of the mammalian protein. This is due only in part to rapid dissociation of iminoacid product and is ascribed to intrinsic differences between the charge-transfer complexes of reduced enzyme flavin and product of the yeast as compared to the mammalian enzyme. With the Trigonopsis enzyme the flavin radical anion appears to be strongly stabilized and can be produced quantitatively.
|(1992): Studies on the active centre of Rhodotorula gracilis D-amino acid oxidase and comparison with pig kidney enzyme Biochemical Journal. 1992, 286(2), pp. 389-394. ISSN 0264-6021. eISSN 1470-8728. Available under: doi: 10.1042/bj2860389||
Studies on the active centre of Rhodotorula gracilis D-amino acid oxidase and comparison with pig kidney enzyme
D-Amino acid oxidase (EC 18.104.22.168) from Rhodotorula gracilis has been reconstituted with 8-chloro-, 8-mercapto-, 6-hydroxy-, 2-thio-, 5-deaza- and 1-deaza-FAD, and the properties of the resulting complexes have been studied and compared with those of the correspondingly modified pig kidney D-amino acid oxidases. Binding appears to be tight for most analogues, at least as tight as for native FAD (~10(-8) M). 8-Mercapto- and 6-hydroxy-FAD bind in their para- and ortho-quinoid forms respectively to yeast D-amino acid oxidase, inferring the presence of a positive charge near the flavin N(1) position, as in the case of the mammalian enzyme. On the other hand, important differences in active-site microenvironment emerge: solvent accessibility to flavin position 8 is drastically restricted in yeast D-amino acid oxidase as indicated by the unreactivity of 8-chloro- and 8-mercapto-FAD enzyme with thiolates and alkylating agents. Significantly different microenvironments are also likely to occur around the flavin positions N(1)-C(2) = 0, N(3)-H and N(5). This is deduced from the differences in interaction of the two proteins with 1-deaza-FAD, 5-deaza-FAD and 2-thio-FAD and from the properties of the respective complexes. The same re-side flavin stereospecificity as shown by the mammalian enzyme was determined for the yeast enzyme using 8-hydroxy-5-deaza-FAD. Thus we can deduce the presence of a similar pattern of functional groups at the active centres of the two enzymes, while the fine tuning of specificity and regulation correlate with environmental differences at specific flavin loci.
|(1991): L-Lactate oxidase MÜLLER, Franz, ed.. Chemistry and biochemistry of flavoenzymes, Vol II. Boca Raton: CRC Press, 1991, pp. 243-289||
dc.contributor.author: Ghisla, Sandro; Massey, Vincent
|Ausschuss für Forschungsfragen||3-1/75|
|Laufzeit:||01.01.1975 – 31.12.2009|