Effects of substitution of tryptophan 412 in the substrate activation pathway of yeast pyruvate decarboxylase

Oligonucleotide-directed site-specific mutagenesis was carried out on pyruv ate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at W412, locat ed on the putative substrate activation pathway and linking E91 on the alph a domain with W412 on the gamma domain of the enzyme. While C221 on the bet a domain is the residue at which substrate activation is triggered [Baburin a, I., et al. (1994) Biochemistry 33, 5630-5635; Baburina, I., et al. (1996 ) Biochemistry 35, 10249-10255], that information, via the substrate bound at C221, is transmitted to H92 on the alpha domain, across the domain divid e from C221 [Baburina, I., et al. (1998) Biochemistry 37, 1235-1244; Baburi na, I., et al. (1998) Biochemistry 37, 1245-1255], thence to E91 on the alp ha domain [Li, H., and Jordan, F. (1999) Biochemistry, 38, 9992-10003], and then on to W412 on the gamma domain and to the active site thiamin diphosp hate located at the interface of the alpha and gamma domains [Arjunan, D., et al. (1996) J. Mel. Biol. 256, 590-600]. Substitution at W412 with F and A was carried out, resulting in active enzymes with specific activities abo ut 4- and 10-fold lower than that of the wild-type enzyme. Even though W412 interacts with E91 and H115 via a main chain hydrogen bond donor and accep tor, respectively, there is clear evidence for the importance of the indole side chain of W412 from a variety of experiments: thermostability, fluores cence quenching, and the binding constants of the thiamin diphosphate, and circular dichroism spectroscopy, in addition to conventional steady-state k inetic measurements. While the substrate activation is still prominent in t he W412F variant, its level is very much reduced in the W412A variant, sign aling that the size of the side chain is also important in positioning the amino acids surrounding the active center to achieve substrate activation. The fluorescence studies demonstrate that W412 is a relatively minor contri butor to the well-documented fluorescence of apopyruvate decarboxylase in i ts native state. The information about the W412 variants provides strong ad ditional support for the putative substrate activation pathway from C221 -- > H92 --> E91 --> W412 --> G413 --> thiamin diphosphate. The accumulating e vidence for the central role of the beta domain in stabilizing the overall structure is summarized.