Site-directed alteration of three active-site residues of a pyruvoyl-dependent histidine decarboxylase.
Site-directed alteration of three active-site residues of a pyruvoyl-dependent histidine decarboxylase.
复制标题
丙酮酰依赖性组氨酸脱羧酶的三个活性位点残基的定点改变。
DOI：
10.1021/bi00069a032
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Robertus,JD
中科院分区：
文献类型：
--
作者：
Pishko,EJ;Robertus,JD
Revised Manuscript Received February 4, 1993 abstract: The active site of histidine decarboxylase (HDC) from Lactobacillus 30a contains a pyruvoyl cofactor sitting at the interface of two molecules in a trimer. Although exhibiting hyperbolic kinetics at pH 4.8, near its optimum, HDC is cooperative at pH 7.6, indicating that the units of the trimer communicate. A Hill plot analysis shows that HDC, at pH 7.6, can be describedby a two-state model. The tense (T) state has an apparent Km for histidine of 50 mM, while the relaxed (R) state has a Km of 5 mM. To explore the catalytic mechanism, three of the cross-boundary active-site residues were altered by site-directed mutagenesis and their effects observed. Ile-59 is known to act as lid on the substrate binding pocket; it was converted to Ala (I59A) and to Val (I59V). The former was inactive, attesting to the importance of this residue in the mechanism. The I59V mutant showed a decrease in Km and in kcat at pHs 4.8 and 7.6. Ile-59 appears to help orient substrate properly for catalysis; decreasing its size expands the binding site. This may allow the substrate to bind more readily, but in a number of conformations which are not optimal for catalysis. Conversion of Tyr-62 to Phe (Y62F) had no effect on catalysis but raised the Km 7-fold at pH 4.8. Asp-63 appears to form an ion pair to the substrate imidazolium. Conversionto the neutral amide (D63N) had no effect on the kcat, but raised the Km 240-fold at pH 4.8. This is consistent with the notion that the ion pair is up to 3 kcal/mol stronger than a simple hydrogen bond with the substrate. The mutant had no detectable activity at pH 7.6.Lactobacillus 30a, induced by histidine, expresses prohistidine decarboxylase (proHDC). This 310-residue proenzyme (chain) is activated by a serinolysis reaction which cleaves the peptide chain between Ser-81 and Ser-82, creating an 81-residue 0 chain and a larger a chain. This process produces a pyruvoyl moiety at the amino terminus of the a chain which serves as the enzyme cofactor for conversion of histidine to histamine and C02. The mechanism of decarboxylation involves Schiff base formation between the substrate and pyruvoyl group (Recsei & Snell, 1970). The history and basic biochemistry of this enzyme have been exhaustively reviewed by van Poelje and Snell (1990). The X-ray structure of activated histidinedecarboxylase (HDC) has been solved to 3.0-Á resolution (Parks et al., 1985) and refined to 2.5-Á resolution (Gallagher et al., 1989). It shows that three HDC molecules trimerize, forming a central well with three active sites near the bottom. Two trimers can also form weak tail-to-tail interactions to form a hexamer (Hackert et al., 1981), but it is unlikely that this has any catalytic significance. The crystallographic model has allowed identification of a number of putative active-site/activation-site residues (Hackert et al., 1987; Gallagher et al., 1989). One important finding is that each active site is formed at the boundary of two molecules related by the molecular3-fold axis. An active site formally located on molecule A will contain the pyruvoyl moiety and a number of amino acid groups such as Glu-197 and Lys-155 from that molecule. In addition, a number of residues from the 0 chain of neighboring molecule B will contribute to that active site. These include residues Ile-59, Tyr-62, Asp-63, and Glu-66 which we will refer to as “cross boundary” residues. In fact, all of these residues lie
