Substrate-cofactor interactions for glycogen phosphorylase b: a binding study in the crystal with heptenitol and heptulose 2-phosphate

Abstract

The structural relationships between substrate and pyridoxal phosphate in glycogen phosphorylase b (EC 2.4.1.1) have been studied by X-ray diffraction experiments at 3-A resolution. Recent work [Klein, H. W., Im, M. J., & Helmreich, E. J. M. (1984) in Chemical and Biological Aspects of Vitamin B6 Catalysis (Evangelopoulos, A. E., Ed.) pp 147-160, Liss, New York] has shown that phosphorylase in the presence of inorganic phosphate catalyzes the conversion of heptenitol to heptulose 2-phosphate. The latter compound is a dead-end product and a most potent inhibitor (Ki = 14 microM). The X-ray diffraction studies show that heptenitol binds at the catalytic site of phosphorylase in a position essentially identical with that observed for the glucopyranose moiety of glucose 1-phosphate. Incubation of a phosphorylase b crystal for 50 h in a solution containing the substrates heptenitol and inorganic phosphate and the activators AMP and maltohetaose resulted in the formation of a phosphorylated product bound at the active site. The structure of this product, as analyzed by a difference Fourier synthesis at 3 A, is consistent with that of heptulose 2-phosphate. Analysis of the surrounding soak solution by thin-layer chromatography showed that heptulose 2-phosphate was produced under these conditions. Heptulose 2-phosphate binds with its glucopyranose moiety in the same position as that for glucose 1-phosphate, but there is a marked difference in phosphate positions. The presence of the methyl group in the beta-configuration in heptulose 2-phosphate forces a change in the torsion angle O5-C1-O1-P from 117 degrees as observe in glucose 1-phosphate to -136 degrees in heptulose 2-phosphate. The "down" position of the phosphate (with respect to the crystallographic z axis) results in a change in the distance between the 5'-phosphorus atom of the pyridoxal phosphate and the phosphorus atom of the substrate from 6.8 (with glucose 1-phosphate) to 4.5 A (with heptulose 2-phosphate). The closest distance between the phosphate oxygen of the cofactor and a phosphate oxygen of heptulose 2-phosphate is 2.7 A, and it is assumed that there must be a hydrogen bond between them. These observations are consistent with the NMR experiments reported in the preceding paper in which sharing of a proton between heptulose 2-phosphate and pyridoxal 5'-phosphate is observed [Klein, H.W., Im, M. J., Palm, D., & Helmreich, E. J. M. (1984) Biochemistry (preceding paper in this issue)].(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple phosphate positions in the catalytic site of glycogen phosphorylase: Structure of the pyridoxal‐5′‐pyrophosphate coenzyme‐substrate analog

“…However, the Glu 672 carboxylate does not appear to be properly oriented to stabilize the glucosyl carbonium ion intermediate that would follow from either the electrophilic or proton-transfer mechanisms. Indeed, as has been noted earlier (McLaughlin et al, 1984;Johnson et al, 1990), there is no residue in the catalytic site of the PLPP-GPb enzyme positioned to stabilize a putative carbonium ion intermediate, suggesting, as proposed by Klein et al (1986) and Palm et al (1990), that the substrate phosphate itself performs that function as a mobile anion. However, it should be noted that no oligosaccharide is present in this structure, and it is well known (Cohn & Cori, 1948) that no half reactions with the natural substrate occur in the absence of oligosaccharide.…”

“…The glucosyl phosphate binding site can be modeled by superposition of GCP, a potent competitive inhibitor of G-1-P (Withers et al, 1981a), into the active site of PLPP-GPb. By virtue of its covalent structure, GCP adopts a strained conformation about its 01-P bond, which is similar to that of heptenito1 2-phosphate bound to the catalytic site of GPb (McLaughlin et al, 1984;Johnson et al, 1990). Johnson et al (1990) suggest that the strained conformation about the 01-P bond would be predicted by stereoelectronic theory to promote cleavage of the a-glycosidic bond; they propose that the natural substrate, G-1-P, adopts a similar conformation as a reactive intermediate in the catalytic mechanism.…”

“…It is now well established by kinetic, NMR, and crystallographic studies that the PLP phosphate participates in catalysis through a direct, noncovalent contact with the phosphate substrate or the phosphate group of the G-1-P product (Withers et al, 1981b;Takagi et al, 1982;Klein et al, 1982Klein et al, , 1986McLaughlin et al, 1984). Key pieces of evidence included the finding that when PLP is replaced by the coenzyme-substrate analog, PLPP, the resultant inactive enzyme is locked into an R-state conformation in the presence of AMP (Withers et al, 1982b).…”

Control of phosphorylase b conformation by a modified cofactor: Crystallographic studies on R‐state glycogen phosphorylase reconstituted with pyridoxal 5′‐diphosphate

“…The position of the P2 phosphate in the PLPP structure is 4.8 A from the position observed for the phosphate of glucose-1-P. In the analysis of the T-state structure the most informative results concerning the mechanism were obtained with the small molecular weight substrate heptenitol and its conversion in the crystal to heptulose-2-phosphate (McLaughlin et al, 1984;Johnson et al, 1990). The structure of the heptulose-2-P complex with G P b in the T state was transformed to superimpose on the R-state structure.…”

Excursions in biophysics by a classical enzymologist

“…Among the reactions of phosphorylase with glucals studied by us, the reaction with D-ghco-heptenitol (Hehre et al, 1980) proved to be especially important because it turned out to proceed only in one direction, e.g., glucosyl transfer to Pi yielding heptulose 2-phosphate. The fact that heptulose 2-phosphate formed from heptenitol is a "suicide" inhibitor that binds with high affinity (K, = 2-14 pM) and in the absence of a primer made it a welcome tool for the X-ray crystallographers (McLaughlin et al, 1984;. Thanks to the formation of heptulose 2-phosphate in the crystal, interaction of the phosphate of the cofactor with the substrate Pi could be demonstrated, which was consistent with the proximity of the phosphates already made apparent by Neal Madsen's and Toshio Fukui's work E.J.M.…”