Kinetic and Crystallographic Studies on the Active Site Arg289Lys Mutant of Flavocytochrome b2 (Yeast L-Lactate Dehydrogenase)

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• 作者：Christopher G. Mowat ; Isabelle Beaudoin ; Rosemary C. E. Durley ; John D. Barton ; Andrew D. Pike ; Zhi-wei Chen ; Graeme A. Reid ; Stephen K. Chapman ; F. Scott Mathews ; and Florence Lederer
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：March 28, 2000
• 年：2000
• 卷：39
• 期：12
• 页码：3266 - 3275
• 全文大小：268K
• 年卷期：v.39,no.12(March 28, 2000)
• ISSN：1520-4995

文摘

Flavocytochrome b<SUB>2 from Saccharomyces cerevisiae couples L-lactate dehydrogenation tocytochrome c reduction. The crystal structure of the native yeast enzyme has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 837-863] as well as that of the sulfite adduct of therecombinant enzyme produced in Escherichia coli [Tegoni, M., and Cambillau, C. (1994) Protein Sci. 3,303-313]; several key active site residues were identified. In the sulfite adduct crystal structure, Arg289adopts two alternative conformations. In one of them, its side chain is stacked against that of Arg376,which interacts with the substrate; in the second orientation, the R289 side chain points toward the activesite. This residue has now been mutated to lysine and the mutant enzyme, R289K-b₂, characterizedkinetically. Under steady-state conditions, kinetic parameters (including the deuterium kinetic isotopeeffect) indicate the mutation affects k_cat by a factor of about 10 and k_cat/K_M by up to nearly 10². Pre-steady-state kinetic analysis of flavin and heme reduction by lactate demonstrates that the latter is entirelylimited by flavin reduction. Inhibition studies on R289K-b₂ with a range of compounds show a generalrise in K_i values relative to that of wild-type enzyme, in line with the elevation of the K_M for L-lactate inR289K-b₂; they also show a change in the pattern of inhibition by pyruvate and oxalate, as well as a lossof the inhibition by excess substrate. Altogether, the kinetic studies indicate that the mutation has alteredthe first step of the catalytic cycle, namely, flavin reduction; they suggest that R289 plays a role both inMichaelis complex and transition-state stabilization, as well as in ligand binding to the active site whenthe flavin is in the semiquinone state. In addition, it appears that the mutation has not affected electrontransfer from fully reduced flavin to heme, but may have slowed the second intramolecular ET step,namely, transfer from flavin semiquinone to heme b₂. Finally, the X-ray crystal structure of R289K-b₂,with sulfite bound at the active site, has been determined to 2.75 Å resolution. The lysine side chain atposition 289 is well-defined and in an orientation that corresponds approximately to one of the alternativeconformations observed in the structure of the recombinant enzyme-sulfite complex [Tegoni, M., andCambillau, C. (1994) Protein Sci. 3, 303-313]. Comparisons between the R289K-b₂ and wild-typestructures allow the kinetic results to be interpreted in a structural context.