Protein engineering on soybean sterol methyl transferase leads to altered substrate binding and catalysis

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Title: Protein engineering on soybean sterol methyl transferase leads to altered substrate binding and catalysis
Author: Sinha, Archana
Abstract: Sterol methyltransferases (SMTs ) are ubiquitously represented in plants and they can serve as the rate -limiting enzymes in the 24 -alkyl sterol (phytosterol ) pathway . Together these enzymes are capable of converting sterol acceptors with a 24 (25 ) -double bond (cycloartenol , CA ; CI -activity ) or 24 (28 ) -double bond ( (24 )28 -methyleneIophenol , ML ; -C2 -activity ) in the sterol side chain into more that 60 distinct phytosterols in a single plant . Recently , we discovered using the soybean SMT that depending on the nature of the substrate olefin bound to SMT , either one or two catalytic reactions can proceed , concerted or step -wise , to generate the product diversity . To investigate the proposed role of aromatic amino acids that are part of a signature motif in the active site of SMT enzymes - F82YEYGWG88 , Y83L and Y83F mutants were prepared and purified to homogeneity and the steady -state kinetic parameters were determined as described in this laboratory for the wild -type soybean SMT . The Y83L mutant performed much like the wild -type enzyme in terms of substrate acceptability , product distribution and physical property , but differences were detected in Y83F mutant . "When the mutant SMT activities were compared to the native SMT activities in relation to inhibition by 25 - azacycloartenol (transition state analog ) or 26 ,27 -dehydrocycloartenol (mechanism based inhibitor ) , both sets of enzymes were found to be inhibited with equal efficacy , suggesting that the successive C -methylation of the Ä24 bond occurs at the same active center . Based on activity assays performed over the temperature range 15 to 40''C , the activation energy (Eact in KJ /mol ) estimated from the Arrhenius plots were found to be : (i ) wild -type , CA = 49 , ML = 71 ; (ii ) Y83F , CA = 65 , ML = 52 and (iii ) Y83L , CA = 98 , ML =185 . Analysis of the pH dependence of log kcat /Km for the wild -type and two mutants showed different profiles for Ä 24 (25 ) and Ä24 (28 ) -substrates . The results of the mutational and kinetic analyses are interpreted to imply that product diversity catalyzed by the soybean SMT is made possible by the relaxed control over substrate and intermediate conformations resulting from altered cation -ð interactions in the active sites of the mutant enzyme and relates to the different mechanisms catalyzed using different olefin substrates .
URI: http : / /hdl .handle .net /2346 /9977
Date: 2004-12

Citation

Sinha, Archana Protein engineering on soybean sterol methyl transferase leads to altered substrate binding and catalysis. Master's thesis, Texas Tech University. Available electronically from http : / /hdl .handle .net /2346 /9977 .

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