Employing Calorimetric Methods to Determine the Mechanism of the Invertase Maximal Activity Delay

转化酶 蔗糖 化学 水解 果糖 差示扫描量热法 等温滴定量热法 酶动力学 酶分析 生物化学 色谱法 热力学 活动站点 物理
作者
Wai Kin Chan,Benjamin Hicks,Alagammai Kaliappan,Nichola C. Garbett,Lee D. Hansen,Jason Kenealey
出处
期刊:ACS food science & technology [American Chemical Society]
卷期号:1 (1): 60-65 被引量:1
标识
DOI:10.1021/acsfoodscitech.0c00011
摘要

Invertase catalyzes the hydrolysis of sucrose to glucose and fructose. Invertase from Saccharomyces cerevisiae has been used in studies of the enzyme catalytic mechanism for more than a century. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) were used to determine the kinetics and physical characteristics of invertase, respectively. Two subspecies of invertase with unique melting points of ∼57 and ∼65 °C are identified using DSC. The rate of invertase-catalyzed sucrose hydrolysis was measured with ITC. ITC has the distinct advantage of directly measuring the rate of reaction, which makes it easy to determine when the enzyme exhibits maximal activity. ITC revealed a previously unknown significant delay after the injections of sucrose before invertase reaches maximal activity. The delay in maximum activity was decreased by a statistically significant (p > 0.05) 85% by an increase in invertase concentration from 1 to 47 μg/mL. Additionally, increasing the temperature of the reaction mixture from 25 to 55 °C resulted in a 66% decrease in the delay to reach maximum activity. However, increasing the sucrose concentration had no effect of the delay in maximum activity. These data are consistent with a change in the conformational or oligomeric state preceding the enzyme reaching full catalytic activity.

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