Assignment of enzymatic functions to specific regions of the PLP-dependentheme protein cystathionine beta-synthase

Cystathionine beta-synthase is a unique heme protein that catalyzes a pyrid oxal phosphate (or PLP)-dependent beta-replacement reaction. The reaction i nvolves the condensation of serine and homocysteine and constitutes one of the two major avenues for detoxification of homocysteine in mammals. The en zyme is allosterically regulated by S-adenosylmethionine (AdoMet). In this study, we have characterized the kinetic, spectroscopic, and ligand binding properties of a truncated catalytic core of cystathionine beta-synthase ex tending from residues 1 through 408 in which the C-terminal 143 residues ha ve been deleted. This is similar to a natural variant of the protein that h as been described in a homocystinuric patient in which the predicted peptid e is 419 amino acids in length. Truncation leads to the formation of a dime ric enzyme in contrast to the tetrameric organization of the native enzyme. Some of the kinetic properties of the truncated enzyme are different from the full-length form, most notably, significantly higher K(m)s for the two substrates, and loss of activation by AdoMet. This is paralleled by the abs ence of AdoMet binding to the truncated form, whereas four AdoMet molecules bind cooperatively to the full-length tetrameric enzyme with a Kd of 7.4 m u M. Steady-state kinetic analysis indicates that the order of substrate ad dition is important. Thus, preincubation of the enzyme with homocysteine le ads to a 2-fold increase in V-max relative to preincubation of the enzyme w ith serine. Since the intracellular concentration of serine is significantl y greater than that of homocysteine, the physiological significance of this phenomenon needs to be considered. Based on ligand binding studies and hom ology searches with protein sequences in the database, we assign residues 6 8-209 as being important for PLP binding, residues 241-341 for heme binding , and residues 421-469 for AdoMet binding.