Coupling of cobalt-carbon bond homolysis and hydrogen atom abstraction in adenosylcobalamin-dependent glutamate mutase

被引:119
作者
Marsh, ENG [1 ]
Ballou, DP
机构
[1] Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USA
[2] Univ Michigan, Sch Med, Dept Biol Chem, Ann Arbor, MI 48109 USA
关键词
D O I
10.1021/bi980512e
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Adenosylcobalamin-dependent glutamate mutase catalyzes an unusual carbon skeleton rearrangement that proceeds through the formation of free radical intermediates generated by the substrate-induced cleavage of the coenzyme cobalt-carbon bond. The reaction was studied at 10 degrees C with various concentrations of L-glutamate and L-threo-3-methylaspartate and with use of stopped-flow spectroscopy to follow the formation of cob(II)alamin. Either substrate induces rapid formation of cob(II)alamin, which accumulates to account for about 25% of the total enzyme species in the steady state when substrate is saturating. Measurements of the rate constant for the formation of cob(II)alamin demonstrate that the enzyme accelerates the rate of homolysis of the cobalt-carbon bond by at least 10(12)-fold. Very large isotope effects on cob(II)alamin formation, of 28 and 35, are observed with deuterated L-glutamate and deuterated L-threo-3-methylaspartate, respectively. This implies a mechanism in which Co-C bond homolysis is kinetically coupled to substrate hydrogen abstraction. Therefore, adenosyl radical can only be formed as a high-energy intermediate only at very low concentrations on the enzyme. The magnitude of the isotope effects suggests that hydrogen tunneling may play an important role catalysis.
引用
收藏
页码:11864 / 11872
页数:9
相关论文
共 29 条
[1]  
BABIOR BM, 1974, J BIOL CHEM, V249, P4537
[2]  
BAHNSON BJ, 1995, METHOD ENZYMOL, V249, P374
[3]   The Yin-Yang of cobalamin biochemistry [J].
Banerjee, R .
CHEMISTRY & BIOLOGY, 1997, 4 (03) :175-186
[4]  
BARKER HA, 1964, J BIOL CHEM, V239, P3260
[5]  
BUCKEL W, 1996, CHEM SOC REV, P329
[6]  
CHEN HP, 1997, BIOCHEMISTRY-US, V36, P14393
[7]  
CLELAND WW, 1982, CRC CR REV BIOCH MOL, V13, P385
[8]   MECHANISM OF ACTION OF COENZYME-B12 - HYDROGEN TRANSFER IN ISOMERIZATION OF BETA-METHYLASPARTATE TO GLUTAMATE [J].
EAGAR, RG ;
HERBST, MM ;
BARKER, HA ;
BALTIMORE, BG ;
RICHARDS, JH .
BIOCHEMISTRY, 1972, 11 (02) :253-+
[9]  
ESSENBERG MK, 1971, J AM CHEM SOC, V93, P1242
[10]  
FERSHT A, 1985, ENZYME STRUCTURE MEC, P136