Histidine-132 Does Not Stabilize a Distal Water Ligand and Is Not an Important Residue for the Enzyme Activity in Heme Oxygenase-1

详细信息    查看全文

• 作者：Kathryn Mansfield Matera ; Hong Zhou ; Catharina Taiko Migita ; Sarah E. Hobert ; Kazunobu Ishikawa ; Koki Katakura ; Hirotaka Maeshima ; Tadashi Yoshida ; and Masao Ikeda-Saito
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：April 22, 1997
• 年：1997
• 卷：36
• 期：16
• 页码：4909 - 4915
• 全文大小：177K
• 年卷期：v.36,no.16(April 22, 1997)
• ISSN：1520-4995

文摘

Heme oxygenase is a key enzyme in the oxygen-dependentheme catabolism pathway. Inorder to clarify the role of highly conserved His132 in heme oxygenaseisoform-1, we have prepared 30kDa truncated rat heme oxygenase mutants in which His132 has beenreplaced by Ala, Gly, and Ser.The expressed recombinant mutant proteins were isolated ininclusion bodies and were recovered fromthe lysis pellet by dissolution in urea followed by dialysis. Thesolubilized fraction obtained, however,was composed of a mixture of a functional enzyme and an inactivefraction. The inactive fraction wasremoved by Sephadex G-75 gel filtration column chromatography, as iteluted out of the column at thevoid volume. The gel filtration-purified heme oxygenase mutantshave spectroscopic and enzymaticproperties identical to those of wild type. The hemin complex ofthe H132A mutant exhibits a transitionbetween a high-spin acid form and a low-spin alkaline form with apK_a value of 7.6 identical to thatinthe wild-type complex. These results demonstrate that His132 inheme oxygenase does not link to thecoordinated water molecule and is not an essential residue for theenzyme activity. These results are inaccordance with our previous preliminary results [Ito-Maki, M.,Ishikawa, K., Mansfield Matera, K., Sato,M., Ikeda-Saito, M., & Yoshida, T. (1995) Arch. Biochem.Biophys. 317, 253-258] but contradictarecent report that His132 is the distal base linked to the coordinatedwater molecule and an importantresidue for enzyme catalysis [Wilks, A., Ortiz de Montellano, P. R.,Sun, J., & Loehr, T. M. (1996)Biochemistry 35, 930-936]. Prolonged storage of thesolubilized fraction from the inclusion bodies ofthe mutants, H132S in particular, results in an increase in the voidvolume fraction with a concomitantdecrease of the 30 kDa fraction. We infer that His132 plays astructural role in stabilization of the hemeoxygenase protein.