meeting follow up

2002 Milwaukee Section Award Photsynthesis and Phosphoribulokinase Jennifer A. Runquist Medical College of Wisconsin

Friday November 15, 2002 Klemmer's Banquet Center 10401 W. Oklahoma Avenue West Allis, WI

DIRECTIONS

The synthesis of ribulose 1,5-bisphosphate (RuBP) by the enzyme phosphoribulokinase (PRK) is essential for the creation of all new biomass (e.g., sugars, sugar polymers such as cellulose, proteins, lipids) in the biosphere. PRK catalyzes an in-line phosphoryl transfer from magnesium adenosine triphosphate (ATP Mg) to ribulose 5-phosphate forming magnesium adenosine diphosphate and ribulose 1,5-bisphosphate (RuBP), the CO₂ acceptor in Calvin's photosynthetic reductive pentose phosphate cycle. This important function of PRK in CO₂ assimilation means that this enzyme exists in a wide variety of organisms. Structurally, plant and algae PRKs exist as dimers of 40 kDa subunits, whereas bacterial PRKs consist of octamers of 32 kDA subunits. Currently, the DNA sequences (genes) and deduced protein primary structures are available for at least 18 organisms. However, between plant and algae PRKs versus bacterial PRKs, sequence similarity is low, less than 20%. Yet invariant amino acid residues cluster in several well defined continuous stretches suggesting their importance to PRK function. Despite differences in quaternary structure, regulatory mechanism and sequence identity, the available chemical and mutagenic data suggest that the active site of both bacterial and plant PRKs are similar.

Cloned PRK has allowed for large quantities of the enzyme to be generated for both kinetic and structural studies. Our high resolution X-ray structure ( 2.4 Å) of Rhodobacter sphaeroides PRK shows that most of the invariant residues are clustered around the active site or are involved in forming the subunit-subunit interface that is conserved between the plant dimer and bacterial octamer. Mutagenesis studies have allowed us to postulate the catalytic and substrate binding roles of invariant amino acids, which provide various chemical resources such as carboxyl groups, amine groups, imdazole groups, metal ligands, etc. Biophysical solution techniques using fluorescence, NMR and ESR have allowed us to characterize PRK binding sites for ATP Mg and the allosteric activator NADH (nicotinamide adenine dinucleotide). These binding studies assure us that mutant enzymes retain a structure similar to wild-type.

PRK is highly regulated. Plant PRKs are regulated by reversible oxidation/reduction of cysteine sulflydryls, which appears to control access to the active site. Bacterial PRKS are activated by a remote NADH binding site. Both of these diverse activation methods communicate the availability of reducing power to the CO₂ fixation pathway. We located the NADH binding site in R. sphaeroides PRK first using X-ray structural methods. Results of mutatgenesis of arginine residues located at this potential binding site followed by fluorescent NADH binding experiments on mutant enzymes confirmed this location as being the remote NADH binding site.

The strategies described in the talk are the methods by which enzymes are currently studied in order to provide insight into the ability of enzymes to catalyze reactions, increasing rates by many orders of magnitude. Further, an understanding of how the rate of catalysis is regulated by enzymes under many cellular conditions is of fundamental importance to our knowledge base. The structure/function information developed about many enzymes has been subsequently utilized to develope drugs and herbicides or been used to understand and potentially modulate and regulate metabolism.

Jennifer A. Runquist is currently a Senior Research Scientist at the Medical College of WI in the Biochemistry Department, where she started as an Assistant Research Professor in 1991. Her area of research interest lies with enzyme mechanisms and enzyme structure. After receiving a B.A. in biochemistry from Oberlin College, Oberlin, Ohio and a Ph.D. in biochemistry from the Chemistry Department of Northwestern University, Evanston, Illinois, she did postdoctoral training at Johns Hopkins University in the Department of Biological Chemistry located in the Medical School. In the Milwaukee area, Jennifer has taught biochemistry at Marquette University and both biochemistry and chemistry at the University of Wisconsin-Milwaukee. Her research interest at UW-Milwaukee involved the enzyme Carbon-Monoxide Dehydrogenase, part of an anaerobic CO₂-fixation pathway.

Jennifer's community service includes initiation of Women in Science of S.E. WI, a group for which she was Vice-President and Program Chair for many years. Additionally, she served on the Milwaukee County Local Emergency Planning Committee (LEPCMC), an official Milwaukee County Board and State Emergency Response Commission Appointment, from 1987 to 1998. This group implemented the Federal Law EPCRA, Emergency Planning and Community-Right-To-Know Act. From 1990-1998 as Chair of the Community Right-To-Know Subcommittee she oversaw hundreds of submissions by all facilities using hazardous chemicals in Milwaukee County. Currently, she is Chair of the Natural Resources Committee of the League of Women Voters Milwaukee County (LWVMC), a committee she has served on since 1985. This committee has been very active in pesticide use policy, options for energy production and use, clean water, clean air, transportation options, hazardous waste, Brownfields reclamation, recycling, Community-Right-To-Know, wetlands protection, and protection of the Great Lakes Ecosystem.

Jennifer is a member of the Division of Biological Chemistry of the American Chemical Society and her involvement with the Milwaukee Section of the ACS includes Editor of the Amalgamator, Chair of the Environmental and Regulatory Affairs Committee, Chair-Elect in 1997, Chair of the Section in 1998, and Past Chair and Chair of the Milwaukee Section Award Committee in 1999.

Jennifer receives her award from Tom McLinn	Jennifer giving her talk
Milwaukee Section Award Winners who were attending the celebration at the November meeting. (l-r) Alex Hill (2000), Norman Hoffman (1983), Sten Flashinski (1995), Jennifer Runquist (2000), Jim Cook (1989), Dan Bloch (1990), Dimitri Gorjestani (1988), Jung-Ja Kim (1999) and Glen Svoboda (1978).