Snider Mark J, Lazarevic Danijela, Wolfenden Richard
Department of Biochemistry & Biophysics and Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.
Biochemistry. 2002 Mar 26;41(12):3925-30. doi: 10.1021/bi011696r.
In neutral solution, 5,6-dihydrocytidine undergoes spontaneous deamination (k25 approximately 3.2 x 10(-5) s(-1)) much more rapidly than does cytidine (k25 approximately 3.0 x 10(-10) s(-1)), with a more favorable enthalpy of activation (DeltaDeltaH# = -8.7 kcal/mol) compensated by a less favorable entropy of activation (TDeltaDeltaS# = -1.8 kcal/mol at 25 degrees C). E. coli cytidine deaminase enhances the rate of deamination of 5,6-dihydrocytidine (kcat/k(non) = 4.4 x 10(5)) by enhancing the entropy of activation (DeltaDeltaH# = 0 kcal/mol; TDeltaDeltaS# = +7.6 kcal/mol, at 25 degrees C). Binding of the competitive inhibitor 3,4,5,6-tetrahydrouridine (THU), a stable analogue of 5,6-dihydrocytidine in the transition state for its deamination, is accompanied by a release of enthalpy (DeltaH = -7.1 kcal/mol, TDeltaDeltaS = +2.2 kcal/mol) that approaches the estimated enthalpy of binding of the actual substrate in the transition state for deamination of 5,6-dihydrocytidine (DeltaH = -8.1 kcal/mol, TDeltaDeltaS = +6.0 kcal/mol). Thus, the shortcomings of THU in capturing all of the binding affinity expected of an ideal transition-state analogue reflect a less favorable entropy of association. That difference may arise from the analogue's inability to displace a water molecule from the "leaving group site" at which ammonia is generated in the normal reaction. The effect on binding of removing the 4-OH group from the transition-state analogue THU, to form 3,4,5,6-tetrahydrozebularine (THZ) (DeltaDeltaH = -2.1 kcal/mol, TDeltaDeltaS = -4.4 kcal/mol), is mainly entropic, consistent with the inability of THZ to displace water from the "attacking group site". These results are consistent with earlier indications [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364] that site-bound water plays a prominent role in substrate activation and inhibitor binding by cytidine deaminase.
在中性溶液中，5,6 - 二氢胞苷发生自发脱氨反应（25℃时k2约为3.2×10⁻⁵ s⁻¹）的速度比胞苷（25℃时k2约为3.0×10⁻¹⁰ s⁻¹）快得多，其活化焓更有利（ΔΔH# = - 8.7 kcal/mol），但被活化熵较不利（25℃时TΔΔS# = - 1.8 kcal/mol）所抵消。大肠杆菌胞苷脱氨酶通过提高活化熵（25℃时ΔΔH# = 0 kcal/mol；TΔΔS# = + 7.6 kcal/mol）来提高5,6 - 二氢胞苷的脱氨速率（kcat/k(non) = 4.4×10⁵）。竞争性抑制剂3,4,5,6 - 四氢尿苷（THU）是5,6 - 二氢胞苷脱氨过渡态的稳定类似物，其结合伴随着焓的释放（ΔH = - 7.1 kcal/mol，TΔΔS = + 2.2 kcal/mol），该值接近5,6 - 二氢胞苷脱氨过渡态中实际底物结合焓的估计值（ΔH = - 8.1 kcal/mol，TΔΔS = + 6.0 kcal/mol）。因此，THU在捕获理想过渡态类似物预期的所有结合亲和力方面的不足反映了缔合熵较不利。这种差异可能源于该类似物无法从正常反应中产生氨的“离去基团位点”取代水分子。从过渡态类似物THU上去除4 - OH基团以形成3,4,5,6 - 四氢泽布勒林（THZ）对结合的影响（ΔΔH = - 2.1 kcal/mol，TΔΔS = - 4.4 kcal/mol）主要是熵效应，这与THZ无法从“进攻基团位点”取代水一致。这些结果与早期的研究结果[Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364]一致，即结合在位点上的水在胞苷脱氨酶的底物活化和抑制剂结合中起重要作用。
