Chen R, Gorenstein D G, Kennedy W P, Lowe G, Nurse D, Schultz R M
Biochemistry. 1979 Mar 6;18(5):921-6. doi: 10.1021/bi00572a030.
N-Acetyl-L-phenylalaninal exists predominantly in its hydrated form in aqueous solution, but the aldehyde and not the hydrate is shown by nuclear magnetic resonance (NMR) spectroscopy to be the effective inhibitor of alpha-chymotrypsin. NMR spectroscopy also indicates that the initial alpha-chymotrypsin-N-acetyl-L-phenylalaninal complex is in equilibrium with a hemiacetal formed between the aldehyde and the active site serine residue. The rate of the latter equilibration is slow on the NMR time scale but the hemiacetal can be detected by cross-saturation NMR spectroscopy. N-Benzoyl-L-phenylalaninal is a more potent inhibitor of alpha-chymotrypsin than the N-acetyl derivative and both the formation of the enzyme-inhibitor complex and the hemiacetal are slow on the NMR time scale, but the hemiacetal in the enzyme can be detected by cross-saturation NMR spectroscopy. The N-acyl-L-phenylalaninals also bind to N-methylhistidinyl-57-alpha-chymotrypsin, but clear evidence for hemiacetal formation was not obtained by cross-saturation NMR spectroscopy either because the hemiacetal was not formed or more probably because the rate of dissociation was slow compared with the rate of relaxation of the hemiacetal proton. The dissociation constant of N-benzoyl-L-phenylalaninal to dehydroalaninyl-195-alpha-chymotrypsin was found to be high relative to the dissociation constant to native alpha-chymotrypsin, supporting the NMR evidence that a hemiacetal with the Ser-195 is formed on association of N-benzoyl-L-phenylalaninal with alpha-chymotrypsin.
N-乙酰-L-苯丙氨醛在水溶液中主要以水合形式存在，但核磁共振（NMR）光谱显示，起作用的α-胰凝乳蛋白酶抑制剂是醛而非水合物。NMR光谱还表明，最初的α-胰凝乳蛋白酶-N-乙酰-L-苯丙氨醛复合物与醛和活性位点丝氨酸残基之间形成的半缩醛处于平衡状态。在NMR时间尺度上，后者的平衡速率较慢，但半缩醛可通过交叉饱和NMR光谱检测到。N-苯甲酰-L-苯丙氨醛是比N-乙酰衍生物更有效的α-胰凝乳蛋白酶抑制剂，在NMR时间尺度上，酶-抑制剂复合物和半缩醛的形成都很缓慢，但酶中的半缩醛可通过交叉饱和NMR光谱检测到。N-酰基-L-苯丙氨醛也与N-甲基组氨酰-57-α-胰凝乳蛋白酶结合，但通过交叉饱和NMR光谱未获得半缩醛形成的确切证据，这要么是因为未形成半缩醛，要么更可能是因为与半缩醛质子的弛豫速率相比，解离速率较慢。相对于天然α-胰凝乳蛋白酶的解离常数，发现N-苯甲酰-L-苯丙氨醛与脱氢丙氨酰-195-α-胰凝乳蛋白酶的解离常数较高，这支持了NMR证据，即N-苯甲酰-L-苯丙氨醛与α-胰凝乳蛋白酶结合时会与Ser-195形成半缩醛。
