Primary and Quaternary Structure

Tryptophanase has been purified from various species, including E. coli,26) Bacillus alvei,7) Aeromortas liquefaciens,i 9) and Proteus rettgeri.l0,M) Preparations of tryptophanase from different sources are similar in molecular mass (about 210 kDa), quaternary structure 

Nucleotide sequence and deduced amino acid sequence of tryptophanase from E coli B/lt7-A and K-12. (Reproduced with permission from Tokushige et al., Biochimie., 71, 714 (1989)). 

Structural genes of tryptophanase from E. coli K-1215 and B/ It7-A16 were cloned and the primary structure deduced as shown in Fig. 9.1. The structure deduced from the nucleotide sequence agrees well with the protein sequence data.I7) Although 29 differences in the nucleotide sequence were found between the tnaA genes of E. coli K-12 and B/ It7-A, the deduced amino acid sequences are completely identical. The total number of amino acid residues is 471 and the molecular mass of a subunit is 52,242 Da. 

The circular dichroism spectrum of E. coli tryptophanase18 showed that the secondary structure of this enzyme seems to be predominantly a-helical. The a-helix content was estimated to be about 50% (Y. Kawata, unpublished results) by the method of Greenfield and Fasman.19  

Honda and Tokushige reinvestigated the effect of temperature and monovalent cations on the quaternary structure of tryptophanase using the HPLC gel-filtration analysis.28 In contrast to the above-mentioned data, they found that in the absence of K+ or NH4+ ions the tetrameric holoenzyme undergoes dissociation into dimers and inactivation at 5°C. Their results also indicate that formation of the active holoenzyme from the apoenzyme and PLP proceeds in two steps the inactive tetrameric species are first formed then converted to the active species only the second step requires the presence of K+ or NH4+ ions (Fig. 9.3) 

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Although E. coli class I and L. leichmanii class II RNR possess characteristic primary and quaternary structures and distinct metallocofactors, they proceed by a similar mechanism, involving an essential thiyl radical [22]. The function of this radical is to abstract the hydrogen atom at the 3 position of the ribose in order to facilitate the cleavage of the (2 )C-OH bond and the reduction of the ribose by a redox active cysteine pair (Scheme 10-2). After each catalytic cycle this pair now as cystine must be reduced for the next cycle. In class I and II RNRs, this occurs by transthiolation from small proteins (such as thioredoxin) that themselves contain redox-active cysteine pairs. 

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