It is hypothesized that inorganic polyphosphate (poly(P)) served as a primitive source of energy and during the course of evolution, there may have been a transition from poly(P) to ATP. The overall objective of this proposal is to characterize enzymes involved in poly(P) metabolism so as to understand the specific mechanistic and molecular structures that result in the utilization of the high energy phosphoryl bond of poly(Ps). The information obtained will then be applied to the ultimate objective of elucidating the role of poly(P) in vivo. The proposed work will be focused on two such enzymes, polyphosphate/ATP glucokinase (poly(P)/ATP GK) and endopolyphosphatases/Exopolyphosphatases (endo PP/exo PP). the poly(P)/ATP GK utilizes either poly(P) of ATP and both activities are catalyzed by the same protein.
Specific aims of experiments include (a) the isolation and sequencing of peptides at the ATP and poly(P) binding and/or catalytic sites of the enzyme from Propionibacterium shermanii, mycobacterium tuberculosis, and Nocardia minima; (b) determination of the kinetic and catalytic mechanisms and (c) isolation and sequencing of the poly(P)/ATP GK gene of P. shermanii and M. tuberculosis from available genomic libraries. The amino acid sequence of the ATP site will be determined using affinity analogs of ATP like 8-Azido-ATP and the lysine specific reagent, sulfonsuccinimidyl- hydroxyphenyl propionate. The purpose will be to compare these with known ATP sequences of glucokinase and hexokinases from higher life forms so as to determine if there are similarities in the ATP sites which will indicate an evolutionary continuity from ancient to more recent evolutionary forms. The poly(P) site peptides will be isolated using phosphate binding-site direct reagents like azidonitrophenyl pyrophosphate and pyridoxal phosphate. The organisms in the proposed studies are classified in the order Actinomycetales and are considered to be primitive. The poly(P) activity is greater than that of ATP in the older members (e.g., P. shermanii) while in the more recent Nocardia, it is reversed. Comparative structural and mechanistic information on the poly(P) and ATP sites may provide information on how substrate binding sites evolve. Protein sequence deduced from the DNA sequence will enable the comparison of whole protein structures with other glucokinases. Isolation of the poly(P)/ATP GK gene will be from the available lambdagt11 and lambdaEMBL3 genomic libraries. the second major focus will be on the characterization of the endo PP and exo PP to determine (a) if the two activities are the catalytic properties of a single protein; (b) whether different endo PP are present that are specific for certain sizes of poly(P). The final objective will be to demonstrate and identify poly(P) synthesizing enzyme(s) in animal cells. the presence of such enzymes in animal cells will be the first direct demonstration of the involvement of poly(P) in in vivo metabolic functions of mammalian cells.
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