Kappelhoff John C, Liu Su Yi Judy, Dugdale Megan L, Dymianiw Dayna L, Linton Larry R, Huber Reuben E
Department of Biological Sciences, University of Calgary, Calgary, AB, T2N 1N4, Canada.
Protein J. 2009 Feb;28(2):96-103. doi: 10.1007/s10930-009-9168-1.
The values of the rate constants and the associated enthalpies and entropies of enzymes with two catalytic steps can be measured by determining the effects of temperature on the k (cat) values. Practical considerations that should be taken into account when doing this are presented. The narrow temperature range available with enzymes and the sensitivity of pH to temperature mean that special attention to detail must be taken and this study highlights the assiduousness needed. The necessity of conversion of apparent k (cat) to true k (cat) values when assays are done with products having pKa values near to the assay pH is shown and the importance of obtaining sufficient data is emphasized. Reasons that non-linear regression should be used to obtain the estimates of rate constants and activation thermodynamic parameters are given. Other precautions and recommendations are also presented. Results obtained by this method for native beta-galactosidase (E. coli) and for a beta-galactosidase in which a Thr was substituted for Asn-460 were analyzed to demonstrate the valuable mechanistic details of enzymes that can be obtained from studies of this type.
具有两个催化步骤的酶的速率常数以及相关的焓和熵的值，可以通过测定温度对k（cat）值的影响来测量。本文介绍了进行此项操作时应考虑的实际因素。酶可用的温度范围较窄以及pH对温度的敏感性意味着必须特别注意细节，本研究突出了所需的严谨性。文中展示了在使用pKa值接近测定pH的产物进行测定时，将表观k（cat）转换为真实k（cat）值的必要性，并强调了获取足够数据的重要性。给出了应使用非线性回归来获得速率常数和活化热力学参数估计值的原因。还介绍了其他注意事项和建议。分析了用这种方法获得的天然β-半乳糖苷酶（大肠杆菌）以及用苏氨酸取代Asn-460的β-半乳糖苷酶的结果，以证明可从这类研究中获得的酶的有价值的机制细节。
