Biochemistry p. 5066 - 5073 (2010)
Update date:2022-08-16
Topics:
Phillips, Robert S.
Lima, Santiago
Khristoforov, Roman
Sudararaju, Bakthavatsalam
The mechanism of wild-type and R37A mutant Pseudomonas dacunhae aspartate β-decarboxylase (ABDC) was studied by rapid-scanning stopped-flow spectrophotometry. Mixing wild-type ABDC with 50 mM disodium l-Asp resulted in the formation of a 325 nm absorption peak within the dead time of the stopped-flow instrument, likely the ketimine of pyridoxamine 5′-phosphate and oxaloacetate or pyruvate. After consumption of the l-Asp, the 360 nm feature of the resting enzyme was restored. Thus, the 325 nm species is a catalytically competent intermediate. In contrast, mixing wild-type ABDC with the disodium salt of either threo- or erythro-β-hydroxy-dl-Asp at 50 mM resulted in a much slower formation of the 325 nm complex, with an apparent rate constant of ~1 or 0.006 s-1, respectively. When wild-type ABDC is mixed with disodium succinate, a nonreactive analogue of l-Asp, formation of a new peak at 425 nm is observed. The apparent rate constant for formation of the 425 nm band exhibits a hyperbolic dependence on succinate concentration, showing that there is a rapid binding equilibrium, followed by a slower reaction in which the internal aldimine is protonated on the Schiff base N. Hydrostatic pressure shifts the spectrum from the 425 nm form to the 360 nm form, consistent with a conformational change. It is likely that the binding of substrate or analogues induces a conformational change that releases strain in the Lys pyridoxal 5′-phosphate Schiff base and increases the pKa, resulting in protonation of the Schiff base to initiate transaldimination. Mixing of R37A mutant ABDC with 50 mM l-Asp also results in the formation of the 325 nm complex, but with an apparent rate constant of 0.2 s-1, at least 5000-fold slower than the rate of wild-type ABDC. In contrast to wild-type ABDC, R37A ABDC shows no change in the cofactor spectrum when mixed with disodium succinate. These results suggest that Arg-37, a conserved active site residue in ABDC, plays a role in modulating the pKa of the pyridoxal 5′-phosphate complexes during catalysis.
View MoreHangzhou J&H Chemical Co., Ltd.
website:http://www.jhechem.com/
Contact:+86-571-87396432
Address:No.200 Zhenhua Rd.Xihu Industrial Park, Hangzhou 310030, China
Sichuan Mianzhu Ronghong Chemical Co.,LTd
Contact:8613981840544
Address:XINSHI INDUSTRY PARK,MIANZHU,SICHUAN,CHINA
Shaanxi King Stone Enterprise Company Limited
Contact:86-29-88353805,13609285751
Address:.209 Keji Road Hi-Tech industrial Develpment Zone .Xian China
Tai zhou world Pharm & Chem Co., Ltd
Contact:+86-576-85301198
Address:Rome 1001,wangjiang plaza,unti 2,jinshan east Road linhai,zhejiang,china
Changzhou Anyi Biochem Co., Ltd.(expird)
Contact:+86-519-88836158
Address:no,51 caoda
Doi:10.1021/ja01628a024
(1955)Doi:10.1021/j100192a074
(1992)Doi:10.1016/j.tetlet.2010.02.139
(2010)Doi:10.1021/jf904490g
(2010)Doi:10.1002/pol.1959.1204013705
(1959)Doi:10.1055/s-1998-1875
(1998)