Battaile K P, Mohsen A W, Vockley J
Department of Medical Genetics, Mayo Clinic, Rochester, Minnesota 55905, USA.
Biochemistry. 1996 Dec 3;35(48):15356-63. doi: 10.1021/bi961113r.
The acyl-CoA dehydrogenases are a family of flavoenzymes with similar structure and function involved in the metabolism of fatty acids and branched chain amino acids. The degree of overlap in substrate specificity is narrow among these enzymes. The position of the catalytic glutamate, identified as Glu376 in porcine medium chain acyl-CoA dehydrogenase (MCAD), Glu254 in human isovaleryl-CoA dehydrogenase (IVD), and Glu261 in human long chain acyl-CoA dehydrogenase (LCAD), has been suggested to affect substrate chain length specificity. In this study, in vitro site-directed mutagenesis was used to investigate the effect of changing the position of the catalytic carboxylate on substrate specificity in short chain acyl-CoA dehydrogenase (SCAD). Glu368, the hypothetical active site catalytic residue of rat SCAD, was replaced with Asp, Gly, Gln, Arg, and Lys and the wild type and mutant SCADs were produced in Escherichia coli and purified. The recombinant wild type SCAD kcat/K(m) values for butyryl-hexanoyl-, and octanoyl-CoA were 220, 22, and 3.2 microM-1 min-1, respectively, while the Glu368Asp mutant gave kcat/K(m) of 81, 12, and 1.4 microM-1 min-1, respectively, for the same substrates. None of the other mutants exhibited enzyme activity. A Glu368Gly/Gly247Glu double mutant enzyme, which places the catalytic residue at a position homologous to that of LCAD, was also synthesized and purified. It showed kcat/K(m) of 9.3, 2.8, and 1.5 microM-1 min-1 with butyryl-, hexanoyl-, and octanoyl-CoA used as substrates, respectively. These results confirm the identity of Glu368 as the catalytic residue of rat SCAD and suggest that alteration of the position of the catalytic carboxylate can modify substrate specificity.
酰基辅酶A脱氢酶是一类黄素酶，其结构和功能相似，参与脂肪酸和支链氨基酸的代谢。这些酶之间底物特异性的重叠程度较窄。催化谷氨酸的位置，在猪中链酰基辅酶A脱氢酶（MCAD）中被鉴定为Glu376，在人异戊酰辅酶A脱氢酶（IVD）中为Glu254，在人长链酰基辅酶A脱氢酶（LCAD）中为Glu261，已被认为会影响底物链长特异性。在本研究中，体外定点诱变被用于研究改变催化羧酸盐位置对短链酰基辅酶A脱氢酶（SCAD）底物特异性的影响。大鼠SCAD的假定活性位点催化残基Glu368被替换为Asp、Gly、Gln、Arg和Lys，并在大肠杆菌中产生野生型和突变型SCAD并进行纯化。重组野生型SCAD对丁酰 - 己酰 - 和辛酰辅酶A的kcat/K(m)值分别为220、22和3.2μM-1 min-1，而Glu368Asp突变体对相同底物的kcat/K(m)分别为81、12和1.4μM-1 min-1。其他突变体均未表现出酶活性。还合成并纯化了一个Glu368Gly/Gly247Glu双突变酶，该酶将催化残基置于与LCAD同源的位置。以丁酰 - 、己酰 - 和辛酰辅酶A为底物时，其kcat/K(m)分别为9.3、2.8和1.5μM-1 min-1。这些结果证实了Glu368是大鼠SCAD的催化残基，并表明催化羧酸盐位置的改变可改变底物特异性。
