(R)-LACTYL-COA DEHYDRATASE FROM CLOSTRIDIUM-PROPIONICUM - STEREOCHEMISTRY OF THE DEHYDRATION OF (R)-2-HYDROXYBUTYRYL-COA TO CROTONYL-COA

被引:32
作者
HOFMEISTER, AEM [1 ]
BUCKEL, W [1 ]
机构
[1] UNIV MARBURG,FACHBEREICH BIOL,MIKROBIOL LAB,KARL VON FRISCH STR,W-3550 MARBURG,GERMANY
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1992年 / 206卷 / 02期
关键词
D O I
10.1111/j.1432-1033.1992.tb16958.x
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
1. A new two-step method for purifying component E II of lactyl-CoA dehydratase was developed. The source of the enzyme was Clostridium propionicum grown on either D,L-alanine or L-threonine. No difference in these preparations was observed whether during purification or by SDS/PAGE of the pure enzymes. Both preparations exhibited similar activities towards (R)-lactyl-CoA as well as towards (R)-2-hydroxybutyryl-CoA, the latter being the superior substrate. 2. Three species of (2R)-2-hydroxybutyrate labelled with H-3 at C3 were prepared containing 96%, 37% and 63% of the H-3 in the 3S-position. By incubation of these species with acetyl-CoA, propionate CoA-transferase and lactyl-CoA dehydratase 104%, 32% and 70% of the H-3, respectively, was released as (HOH)-H-3. The data indicate the stereospecific abstraction of the 3Si hydrogen of (2R)-2-hydroxybutyryl-CoA during the dehydration. 3. The identity of the product of the dehydration as crotonyl-CoA was established by the combined action of the enzymes crotonase and (S)-3-hydroxyacyl-CoA dehydrogenase. The results indicate that the elimination of water from (R)-2-hydroxybutyryl-CoA occurs in a syn mode. 4. All enzyme activities necessary for the conversion Of L-threonine via (R)-2-hydroxybutyryl-CoA to butyrate were detected in cell-free extracts of C. propionicum. 5. A new mechanism for the dehydration of lactyl-CoA is proposed.
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页码:547 / 552
页数:6
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