Sulfate activation and transport in mammals: system components and mechanisms

doi:10.1016/S0009-2797(97)00129-4

Chemico-Biological Interactions

Volume 109, Issues 1–3, 20 February 1998, Pages 143-151

https://doi.org/10.1016/S0009-2797(97)00129-4 Get rights and content

Abstract

Extensive studies on the mammalian sulfate-activating enzymes and PAPS translocase have enhanced our understanding of the overall pathway of sulfate activation and utilization. Isolation of the PAPS-synthesizing activities from rat chondrosarcoma and preparation of stable non-hydrolyzable analogs of APS and PAPS have facilitated the kinetic characterization of mammalian ATP sulfurylase and APS kinase. These studies provided the basis for further experimental work showing that APS, the labile intermediate product, is channeled directly between the sulfurylase and kinase active sites. The defect in the brachymorphic mutant mouse lies in this channeling mechanism, thus interfering with efficient PAPS production. The rat chondrosarcoma ATP sulfurylase and APS kinase activities, in fact, reside in a single bifunctional cytoplasmic protein, which has now been cloned and expressed. The mechanism by which PAPS reaches its sites of utilization in the Golgi lumen has also been elucidated: The PAPS translocase is a 230-kDa integral Golgi membrane protein which functions as an antiport.

Introduction

The sulfate activation pathway in mammals consists of two activities, ATP sulfurylase (EC 2.7.7.4) which catalyzes synthesis of APS from ATP and SO₄⁻² and APS kinase (EC 2.7.1.25) which phosphorylates APS in the presence of another molecule of ATP (Fig. 1). The same two activities are involved in sulfate activation in simpler organisms where they appear to be relatively small, separate enzymes. We have previously studied the PAPS-synthesizing enzymes in the context of a defect in the production of PAPS in the brachymorphic mutant mouse, where a reduction in the activities of both enzymes was clearly demonstrated 1, 2, 3. In order to understand this intriguing double enzyme defect, it was necessary to elucidate the relationship between the two activities by first purifying and attempting to determine whether they represented two separate polypeptides or a single bifunctional polypeptide. Toward this end, the two sulfate-activating enzymes, ATP sulfurylase and APS kinase, were each purified from rat chondrosarcoma and shown to have nearly identical molecular properties and fractionation behavior [4]. To further our evidence as to whether a single polypeptide with multiple active sites or two tightly complexed polypeptides pertain, characterization of both kinetic mechanisms, as well as further affinity purification and eventual cloning of the sulfate activation enzymes was accomplished. These studies identified the mammalian sulfurylase/kinase as a bifunctional enzyme that uses a channeling mechanism to transfer the intermediate APS efficiently from the sulfurylase to the kinase active site. The finding of multiple functions on a single polypeptide suggests that this complex enzyme is a critical locus for regulation and a vunerable site for mutations.

In addition, components which are involved in processes distal and proximal to the sulfurylase/kinase complex, thereby constituting the entire sulfate uptake, activation and utilization pathway (Fig. 1), remained unknown with respect to the number and nature of participants, rate-limiting steps and modes of regulation. Maximum efficiency of this overall pathway requires transport of inorganic sulfate into the cell and then transport of the product of the activation pathway, PAPS, from the site of synthesis in the cytosol to the site(s) of utilization in the Golgi lumen. Clearly transport across inter- and intra-cellular membranes is a hallmark of this overall pathway. Thus, another potential site of regulation of the sulfation process relates to the mechanism by which PAPS generated in the cytosol is transferred (or translocated) across the Golgi membrane to intralumenal sites of PAPS utilization. It is hypothesized that this translocation process must be exquisitely coupled to biosynthesis of PAPS for maximum efficiency of the overall process. Thus identification and characterization of the PAPS translocase was necessary for gaining a better understanding of these individual steps as well as the process of sulfation in general.

Section snippets

Synthesis of APS and PAPS analogs

In concert with the purification and molecular characterization, kinetic analysis of the ATP sulfurylase and APS kinase activities individually and in coupled reactions, have contributed to understanding the nature of their relationship to each other. Mechanistic studies of the enzymes involved in synthesis and utilization of APS and PAPS were hampered by the extreme liability of APS and PAPS, the vulnerability of the compounds to enzymatic degradation, the unfavorable equilibrium constant for

Mechanistic studies of PAPS enzymes

Based on our early work showing that the brachymorphic mouse exhibits a dual defect affecting both PAPS enzymes 1, 2, 3and then showing that they fractionate together through extensive purification and exhibit several common properties and behavior [4], additional strategies for exploring their relationship were sought. Since defects in consecutive activities of a metabolic sequence are uncommon in mammals, such observations suggest ways in which single abnormalities may alter both enzymes.

Intermediate channeling between ATP sulfurylase and APS kinase

Three types of experiments were used to examine the relationship of these two sequential activities from rat chondrosarcoma. In the first, an assay system that permits measuring the accumulation of both APS and PAPS in the presence of both enzyme activities yielded a PAPS/APS ratio corresponding to a channeling efficiency of 96%. Second, the velocity of the APS kinase reaction measured in the overall system, utilizing endogenously synthesized APS, was 8-fold greater than that of the isolated

ATP sulfurylase and APS kinase reside on a single bifunctional protein

As mentioned, both sulfurylase and kinase from rat chondrosarcoma co-purified over 2000-fold through S300 gel filtration, hydroxylapatite and ATP affinity chromatography, suggesting the activities are inseparable [4]. In subsequent studies both activities co-purified through substrate affinity chromatography using stable analogs of APS and PAPS. Approximately 56-kDa protein, containing both activities, was purified to apparent homogeneity through reversed-phase chromatography and observed on

Identification and characterization of PAPS translocase

Our interest in overall synthesis and utilization of the universal high energy sulfate donor, PAPS, also extends to the process by which the PAPS generated in the cytosol is transferred across the Golgi membrane to intralumenal sites of PAPS utilitization (Fig. 1). Toward this end, we found that the protein responsible for the PAPS translocating activity could be solubilized from vesicles enriched in enzyme markers for the Golgi apparatus and reconstituted into liposomes [13]. In order to

Acknowledgements

This work was supported by NIH grants HD-17332 and AR-19622.

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Sulfate activation and transport in mammals: system components and mechanisms

Abstract

Introduction

Section snippets

Synthesis of APS and PAPS analogs

Mechanistic studies of PAPS enzymes

Intermediate channeling between ATP sulfurylase and APS kinase

ATP sulfurylase and APS kinase reside on a single bifunctional protein

Identification and characterization of PAPS translocase

Acknowledgements

Arch. Biochem. Biophys.

J. Biol. Chem.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.