The Active Site Loop of S-Adenosylmethionine Synthetase Modulates Catalytic Efficiency

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• 作者：John C. Taylor ; Fusao Takusagawa ; and George D. Markham
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：July 30, 2002
• 年：2002
• 卷：41
• 期：30
• 页码：9358 - 9369
• 全文大小：274K
• 年卷期：v.41,no.30(July 30, 2002)
• ISSN：1520-4995

文摘

Crystallographic studies of Escherichia coli S-adenosylmethionine synthetase (ATP:L-methionineS-adenosyltransferase, MAT) have defined a flexible polypeptide loop that can gate access to the activesite without contacting the substrates. The influence of the length and sequence of this active site loop oncatalytic efficiency has been characterized in a mutant in which the E. coli MAT sequence (DRADPLEQ)has been replaced with the distinct sequence of the corresponding region of the otherwise highly homologousrat liver enzyme (HDLRNEEDV). Four additional mutants in which the entire DRADPLEQ sequencewas replaced by five, six, seven, or eight glycines have been studied to unveil the effects of loop lengthand the influence of side chains. In all of the mutants, the maximal rate of S-adenosylmethionine formation(k_cat) is diminished by more than 200-fold whereas the rate of hydrolysis of the tripolyphosphate intermediateis decreased by less than 3-fold. Thus, the function of the loop is localized to the first step in the overallreaction. The K_m for methionine increases in all of the oligoglycine mutants, whereas the K_m values forATP are not substantially different. The k_cat for the wild-type enzyme is decreased by increases in solutionmicroviscosity with 55% of the maximal dependence. Thus, a diffusional event is coupled to the chemicalstep of AdoMet formation, which is known to be rate-limiting. The results indicate that a conformationalchange, possibly loop closure, is associated with AdoMet synthesis. The data integrate a previouslydiscovered conformational change associated with PPP_i binding to the E·AdoMet complex into the reactionsequence, reflecting a difference in protein conformation in the E·AdoMet·PPP_i complex whether it isformed from the E·ATP·methionine complex or from binding of exogenous PPP_i. The temperaturedependence of the k_cat for S-adenosylmethionine formation shows that the removal of the side chains inthe glycine mutants causes the activation enthalpy of the reaction to approximately double in each case,while the activation entropy changes from negative in the wild-type enzyme to positive in the mutants.The favorable activation entropy in the mutant-catalyzed reactions may reflect release of water duringcatalysis, while the negative activation entropy in the reaction catalyzed by the wild-type enzyme apparentlyreflects reorganization of the loop. The observations point to how nature can fine-tune the activity of anenzyme by modifying substrate and product access to the active site rather than by altering the enzyme·substrate contacts or the catalytic machinery itself.